Novel Lactobacillus Having Various Functions, and Use Thereof

ABSTRACT

The present invention provides novel Lactobacillus sp. strains, novel Bifidobacterium sp. strains, or lactic acid bacteria mixtures thereof, which are isolated from kimchi or human feces. A certain Lactobacillus sp. strain or certain Bifidobacterium sp. strain according to the present invention is isolated from kimchi or human feces, and thus is highly safe, and has various physiological activities such as antioxidant activity, β-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity or tight junction protein expression-inducing activity. Accordingly, a certain Lactobacillus sp. strain, certain Bifidobacterium sp. strain or mixture thereof according to the present invention may be used as a functional food or medicinal material useful for the prevention, alleviation or treatment of intestinal damage, liver injury, allergic disease, inflammatory disease, or obesity.

TECHNICAL FIELD

The present invention relates to novel lactic acid bacteria and thelike, and more particularly to novel lactic acid bacteria or novellactic acid bacteria mixtures, which are isolated from kimchi or humanfeces and have various physiological activities such as antioxidantactivity, β-glucuronidase-inhibitory activity, lipopolysaccharide (LPS)production-inhibitory activity or tight junction proteinexpression-inducing activity. Moreover, the present invention relates tovarious food and medicinal uses of novel lactic acid bacteria or novellactic acid bacteria mixtures.

BACKGROUND ART

As humanity has developed into a prosperous society, the fast food hasbeen rapidly become popular and the pattern of diseases has also changeddramatically. In particular, in modern people, intestinal floradisturbance, intestinal permeability syndrome, colitis, liver diseases,allergic diseases, obesity and the like are increasing due to fast foodeating habits based on meat and fat, irregular meal, excessive drinking,lack of exercise, excessive stress, exposure to harmful environments,and the like.

Intestinal Flora Disturbance

There are many bacteria living in the gastrointestinal tract of thehuman body. The human body has about 10 trillion normal cells, but hasabout 100 trillion bacteria which are about 10-fold larger than thenormal cells. These bacteria can be divided into beneficial bacteriathat help human intestinal health and harmful bacteria that are harmfulto human health. The health of human body can be maintained whenbeneficial bacterial such as Lactobacillus, Bifidobacterium,Streptococcus, Leuconostoc, Pediococcus, Sporolactobacillius and thelike are more dominant in the gastrointestinal tract than harmfulbacteria. Otherwise, diseases can be caused, such as obesity, intestinalpermeability syndrome, liver diseases, accelerated aging, enteritis andthe like.

Intestinal Permeability Syndrome

The gastrointestinal tract of the human body is composed of mucus andvilli, which efficiently absorb nutrient components, but prevent theabsorption of pathogenic microorganisms having a high molecular weightor toxins produced by these microorganisms. In addition, the human bodyhas an immune system capable of protecting the body from invasion ofexternal antigens having a high molecular weight. However, due toinfection with many pathogenic microorganisms or toxins, excessivestress, intake of foods such as high-fat diets capable of proliferatingharmful bacterial living in the gastrointestinal tract, excessivealcohol intake, the abuse of drugs (e.g., antibiotics) and the like,intestinal flora is disturbed, abnormalities in the gastrointestinaltract's immune system occur, and expression of tight junction proteinsis inhibited. If expression of tight junction proteins is inhibited,tight junction of intestinal mucosa becomes loosened, and the invasioninto the body of large molecules due to the loosened gap andabnormalities in the immune system.

Intestinal permeability syndrome is also known as leaky gut syndrome,and refers to a condition in which external such as less digested foods,pathogenic microorganisms, toxins or the like are continuouslyintroduced into blood, because the tight junction barrier system ofepithelial cells forming the gastrointestinal tract is not smoothlyoperated. When intestinal permeability syndrome occurs, externalantigens that are generally not absorbed into the body enter the body,thus causing ulcerative colitis, Crohn's disease, liver injury, liverdysfunction, allergic diseases (including asthma), atopy, autoimmunediseases, steatorrhea, digestive absorption disorder, acne, acceleratedaging, endotoxemia, intestinal infection, eczema, irritable bowelsyndrome, chronic fatigue syndrome, psoriasis, rheumatoid arthritis,pancreatic insufficiency, inflammatory joint diseases or the like.

Colitis

Although it was previously known that the incidence of ulcerativecolitis and Crohn's disease is high in Europeans, the number of patientswith ulcerative colitis and Crohn's disease in oriental countriesincluding Korea has recently increased rapidly due to changes inlifestyles such as eating habits. However, the cause is unclear, andthus a fundamental treatment method for these diseases has not yet beenestablished. For this reason, drugs are used which do not aim tocompletely treat, but aim to relieve symptoms and maintain this relievedstate over the longest possible period. As drugs for this symptomatictherapy, aminosalicylic acid agents, adrenocorticosteroid agents,immunosuppressants and the like are mainly used, but have been reportedto cause various side effects. For example, sulfasalazine which isfrequently used as an aminosalicylic acid agent was reported to causeside effects, including nausea, vomiting, anorexia, rash, headache,liver injury, leukocytopenia, abnormal red blood cells, proteinuria,diarrhea and the like. In addition, adrenocorticosteroid agents aregenerally used by prednisolone oral administration, infusion,suppository, intravenous injection or the like, but cause strong sideeffects such as gastric ulcer or femoral necrosis upon long-term use.However, discontinuation of medication can cause symptoms to recur, andthus these drugs must be continuously used. Accordingly, there is a needto develop agents for treating intestinal bowel diseases, such asulcerative colitis, Crohn's diseases and the like, which have excellenteffects, are safe and cause no side effects. Irritable bowel syndrome(IBS) is also a chronic abdominal disease whose cause is unclear.Currently, there is no fundamental therapeutic agent for IBS, andsymptomatic therapy is performed for the purpose of relieving symptomsof each type of IBS. For example, for diarrhea-IBS, an anticholinergicagent having spasmolytic action that suppresses the contraction ofsmooth muscles is used, and for constipation-IBS, salt laxatives areused. For alternating-IBS difficult to control with drugs, an agent forimproving gastrointestinal motor function is fundamentally used.

Liver Diseases

The liver in the human body plays roles such as energy metabolism(nutrient treatment and storage, and waste excretion), detoxification oftoxins, synthesis of serum proteins, and smooth absorption of fat in thebowel by bile juice secretion, and is also important in immunitymaintenance (body defense) and vitamin metabolism. However, infectionwith hepatitis viruses or excessive intake of alcohol or high-fat mealsmay cause liver diseases such as hepatitis, fatty liver or livercirrhosis. In addition, liver diseases may also be caused by drugs(tuberculosis therapeutic drugs, aspirin, antibiotics, anesthetics,antihypertensive drugs, oral contraceptives, etc.), congenital metabolicdisorders, heart failure, shock, or the like. When liver disease occurs,it can develop into chronic hepatitis, starting with acute hepatitiswith fatigue, vomiting, diarrhea, anorexia, jaundice, right upperquadrant pain, fever or muscle pain.

Allergic Diseases

As society has become more complicated and the industry and civilizationhas developed, environmental pollution and stress have increased, and aseating habits have changed, patients with allergic diseases haveincreased every year. Patients with allergic diseases such as atopy,anaxylosis, asthma and the like were less than 1% in 1980, but increasedrapidly to 5% or more in 2000s, and are estimated to be more than 10%,including potential patients. Allergic diseases are caused by excessiveimmune responses of a body, which result from antigen-antibodyreactions, and allergic diseases are classified into types 1 to 4hypersensitivity reactions based on response time and whethercomplements are involved. Type 1 hypersensitivity reactions includeatopy, anaphylactic shock, bronchial asthma, urticaria, pollinosis andthe like; type 2 hypersensitivity reactions include inadequatetransfusion, autoimmune hemolytic anemia, hemolytic anemia caused bydrugs, granulocytopenia, thrombocytopenic purpura and the like; type 3hypersensitivity reactions include erythema, lymphatic swelling,arthralgia, arthritis, nephritis, acute glomerulonephritis followingstreptococcal infection, and the like; and type 4 hypersensitivityreactions include chronic inflammation and the like. To improve allergicdiseases, it is preferable to remove allergens (house dust, mites, etc.)from the skin by showering or bathing and avoid allergen intake.However, when allergic diseases are not improved, drugs such assteroids, antihistamines, immunosuppressants or the like are used, whicheasily cause side effects such as skin atrophy, vasodilation,discoloration, purpura (steroids), drowsiness (antihistamines), kidneyfailure (immunosuppressants) and the like. Among the drugs developed sofar, there is no drug that can completely cure allergies, and thesedrugs are expected to improve symptoms, but have the problem of causingsignificant side effects.

Obesity

Obesity is a metabolic disorder caused by the imbalance of calorieintake and consumption, and is caused by the increased size(hypertrophy) or increased number (hyperplasia) of in vivo adipocytes inmorphological terms. Obesity is not only the most common malnutritiondisorder in western society, and the prevalence of obesity in Korea isalso rapidly increasing due to the improvement of eating habits andwesternization of lifestyles. Therefore, the importance of treatment andprevention of obesity has been greatly emphasized. Obesity is animportant factor that disturbs the individual in psychological terms andalso increases the risk of various adult diseases in social terms.Obesity is known to be directly related to the increased prevalence ofvarious adult diseases such as type 2 diabetes, hypertension,hyperlipidemia, heart disease and the like (Cell 87:377, 1999), anddiseases related to obesity are collectively referred to as metabolicsyndrome or insulin resistance syndrome, and these diseases have beenreported to cause arteriosclerosis and cardiovascular diseases. Obesitytherapeutic agents known so far Xenical (Roche Pharmaceuticals,Switzerland), Reductil (Abbott, USA), Exolise (Arkopharma, France) andthe like, and are largely classified into appetite suppressants, energyexpenditure promoters, and fat absorption inhibitors. Most obesitytherapeutic agents are appetite suppressants that suppress appetite bycontrolling the neurotransmitters associated with the hypothalamus.However, conventional therapeutic agents cause side effects such asheart diseases, respiratory diseases, neurological diseases and thelike, and the persistence of their effects is also low. Thus, thedevelopment of improved obesity therapeutic agents is required. Inaddition, among currently developed products, there are little or notherapeutic agents that have satisfactory therapeutic effect withoutcausing side effects, and thus the development of a new therapeuticagent for obesity is required.

Probiotics are collectively referred to as live microorganisms thatimprove the host's microbial environment in the gastrointestinal tractof animals, including humans, and have beneficial effects on the host'shealth. In order to be effective as probiotics, it is necessary to haveexcellent acid resistance, bile resistance and adherence to epithelialcells, because most of these probiotics should reach the small intestineupon oral administration and must be adhered to the intestinal surface.Lactic acid bacteria are used as probiotics because they play a role indecomposing fibrous and complex proteins to make important nutrientswhile living in the digestive system of the human body. Lactic acidbacteria have been reported to exhibit effects such as maintenance ofintestinal normal flora, improvement of intestinal flora, anti-diabeticand anti-hyperlipidemic effects, inhibition of carcinogenesis,inhibition of colitis, and nonspecific activity of the host's immunesystem. Among these lactic acid bacteria, Lactobacillus sp. strains aremajor members of normal microbial communities living in the bowel of thehuman body and have long been known to be important in maintaining ahealthy digestive tract and vaginal environment. Currently, according tothe U.S. Public Health Service guidelines, all the Lactobacillus strainsdeposited with the American Type Culture Collection (ATCC) areclassified as ‘Bio-Safety Level 1’, which is recognized as having noknown potential risk of causing disease in humans or animals. Meanwhile,lactic acid bacteria of kimchi that are involved in kimchi fermentationhave been reported to have immune enhancement effects, antimicrobialeffects, antioxidant effects, anti-cancer effects, anti-obesity effects,hypertension preventive effects or constipation preventive effects[Hivak P, Odrska J, Ferencik M, Ebringer L, Jahnova E, Mikes Z. One-yearapplication of Probiotic strain Enterococcus facium M-74 decreases Serumcholesterol levels. Bratisl lek Listy 2005; 106(2); 67-72;Agerholm-Larsen L. Bell M L. Grunwald G K. Astrup A.: The effect of aprobiotic milk product on plasma cholesterol: a metaanalysis ofshort-term intervention studies; Eur J Clin Nutr. 2000; 54(11) 856-860;Renato Sousa, Jaroslava Helper, Jian Zhang, Strephen J Lewis and Wani OLi; Effect of Lactobacillus acidophilus supernants on body weight andleptin expression in rats; BMC complementary and alternative medicine.2008; 8(5)1-8].

Since various bioactive activities of lactic acid bacteria were known,studies have recently been conducted to develop lactic acid bacterialstrains that have excellent functions while being safe for the human andto apply these strains to medicines or functional foods. For example,Korean Patent Application Publication No. 10-2009-0116051 disclosesLactobacillus brevis HY7401 having the effects of treating andpreventing colitis. Furthermore, Korean Patent Application PublicationNo. 10-2006-0119045 discloses lactic acid bacteria for preventing ortreating atopic dermatitis, which is selected from the group consistingof Leuconostoc citreum KACC91035, Leuconostoc mesenteroides subsp.mesenteroides KCTC 3100 and Lactobacillus brevis KCTC 3498. Furthermore,Korean Patent Application Publication No. 10-2013-0092182 discloses afunctional health food for preventing alcoholic liver disease orrelieving hangovers, which comprises Lactobacillus brevis HD-01(accession number: KACC91701P) having an excellent ability to decomposealcohol. In addition, Korean Patent Application Publication No.10-2010-0010015 discloses a Lactobacillus johnsonii HFI 108 strain (KCTC11356BP) having blood cholesterol lowering and anti-obesity activities.In addition, Korean Patent Application Publication No. 10-2014-0006509discloses a composition for preventing or treating obesity comprising aBifidobacterium longum CGB-C11 strain (accession number: KCTC 11979BP)that produces conjugated linoleic acid as an active ingredient.

However, there has been no report of lactic acid bacteria-relatedtechnology capable of alleviating or treating all of intestinal floradisturbance, intestinal permeability syndrome, colitis, liver diseases,allergic diseases, obesity and the like, which are increasing in modernhumans. Therefore, there is a need to screen a novel strain havingvarious functionalities and to develop medicines, functional foods andthe like by use of this strain.

DISCLOSURE Technical Problem

The present invention has been made under the Background Art asdescribed above, and it is an object of the present invention to providenovel lactic acid bacteria having various physiological activities orfunctionalities required for probiotics, and the food and medicinal usesthereof.

Another object of the present invention is to provide a novel lacticacid bacteria mixture capable of exhibiting various maximizedphysiological activities or functionalities, and the food and medicinaluses thereof.

Technical Solution

The present inventors have screened numerous lactic acid bacteria fromkimchi or human feces, and have found that, among these screened lacticacid bacteria, a certain Lactobacillus sp. strain, a certainBifidobacterium sp. strain or a mixture thereof has an excellent effecton the improvement of intestinal damage such as intestinal permeabilitysyndrome, liver injury such as fatty liver, allergic diseases such asatopic dermatitis, inflammatory diseases such colitis, or obesity,thereby completing the present invention.

To achieve the above objects, an embodiment of the present inventionprovides a lactic acid bacteria selected from Lactobacillus breviscomprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 1,Bifidobacterium longum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 3, Lactobacillus plantarum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 4, Lactobacillusplantarum comprising a 16S rDNA nucleotide sequence represented by SEQID NO: 5 or Bifidobacterium longum comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 7. The Lactobacillus brevis,Lactobacillus plantarum or Bifidobacterium longum has antioxidantactivity, β-glucuronidase-inhibitory activity, lipopolysaccharide (LPS)production-inhibitory activity or tight junction proteinexpression-inducing activity. Another embodiment of the presentinvention provides a pharmaceutical composition for preventing ortreating intestinal damage, liver injury, allergic disease, inflammatorydisease or obesity comprising lactic acid bacteria selected fromLactobacillus brevis comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 1, Bifidobacterium longum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 3, Lactobacillusplantarum comprising a 16S rDNA nucleotide sequence represented by SEQID NO: 4, Lactobacillus plantarum comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 5 or Bifidobacterium longumcomprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 7, aculture of the lactic acid bacteria, a lysate of the lactic acidbacteria or an extract of the lactic acid bacteria as an activeingredient. Still another embodiment of the present invention provides afood composition for preventing or alleviating intestinal damage, liverinjury, allergic disease, inflammatory disease or obesity comprisingLactobacillus brevis comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 1, Bifidobacterium longum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 3, Lactobacillusplantarum comprising a 16S rDNA nucleotide sequence represented by SEQID NO: 4, Lactobacillus plantarum comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 5 or Bifidobacterium longumcomprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 7, aculture of the lactic acid bacteria, a lysate of the lactic acidbacteria or an extract of the lactic acid bacteria as an activeingredient.

To achieve other objects of the present invention, an embodiment of thepresent invention provides a mixture of two or more lactic acid bacteriaselected from the group consisting of Lactobacillus brevis comprising a16S rDNA nucleotide sequence represented by SEQ ID NO: 1,Bifidobacterium longum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 3, Lactobacillus plantarum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 4, Lactobacillusplantarum comprising a 16S rDNA nucleotide sequence represented by SEQID NO: 5, and Bifidobacterium longum comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 7. The mixture of lactic acidbacteria has antioxidant activity, β-glucuronidase-inhibitory activity,lipopolysaccharide (LPS) production-inhibitory activity or tightjunction protein expression-inducing activity. Another embodiment of thepresent invention provides a composition for preventing or treatingintestinal damage, liver injury, allergic disease, inflammatory diseaseor obesity comprising a mixture of two or more lactic acid bacteriaselected from the group consisting of Lactobacillus brevis comprising a16S rDNA nucleotide sequence represented by SEQ ID NO: 1,Bifidobacterium longum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 3, Lactobacillus plantarum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 4, Lactobacillusplantarum comprising a 16S rDNA nucleotide sequence represented by SEQID NO: 5 and Bifidobacterium longum comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 7, a culture of the mixture of lacticacid bacteria, a lysate of the mixture of lactic acid bacteria or anextract of the mixture of lactic acid bacteria as an active ingredient.Still another embodiment of the present invention provides a foodcomposition for preventing or alleviating intestinal damage, liverinjury, allergic disease, inflammatory disease or obesity comprising amixture of two or more lactic acid bacteria selected from the groupconsisting of Lactobacillus brevis comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 1, Bifidobacterium longum comprisinga 16S rDNA nucleotide sequence represented by SEQ ID NO: 3,Lactobacillus plantarum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 4, Lactobacillus plantarum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 5 and Bifidobacteriumlongum comprising a 16S rDNA nucleotide sequence represented by SEQ IDNO: 7, a culture of the mixture of lactic acid bacteria, a lysate of themixture of lactic acid bacteria, or an extract of the mixture of lacticacid bacteria as an active ingredient.

Advantageous Effects

A certain Lactobacillus sp. strain or certain Bifidobacterium sp. strainaccording to the present invention is isolated from kimchi or humanfeces, and thus is highly safe, and has various physiological activitiessuch as antioxidant activity, β-glucuronidase-inhibitory activity,lipopolysaccharide (LPS) production-inhibitory activity or tightjunction protein expression-inducing activity. Accordingly, a certainLactobacillus sp. strain, certain Bifidobacterium sp. strain or mixturethereof according to the present invention may be used as a functionalfood or medicinal material useful for preventing, alleviating ortreating of intestinal damage, liver injury, allergic disease,inflammatory disease or obesity.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the change in GOT value when lactic acidbacteria were administered to model animals having liver injury inducedby D-galactosamine, in a first experiment of the present invention.

FIG. 2 is a graph showing the change in GPT value when lactic acidbacteria were administered to model animals having liver injury inducedby D-galactosamine, in a first experiment of the present invention.

FIG. 3 is a graph showing the change in MDA value when lactic acidbacteria were administered to model animals having liver injury inducedby D-galactosamine, in a first experiment of the present invention.

FIG. 4 is a graph showing the effect of lactic acid bacteria, screenedin a first experiment of the present invention, on thelipopolysaccharide (LPS)-induced inflammatory response of dendriticcells. The left graph in FIG. 4 shows the effect of lactic acid bacteriaon cells not treated with LPS (lipopolysaccharide), and the right graphshows the effect of lactic acid bacteria on cells treated with LPS(lipopolysaccharide).

FIG. 5 is a graph showing the effect of Bifidobacterium longum CH57 onthe LPS (lipopolysaccharide)-induced inflammatory response ofmacrophages in a first experiment of the present invention.

FIG. 6 shows the results of analyzing the effect of Lactobacillus brevisCH23 on the differentiation of T cells (isolated from spleen) into Th17cells or Treg cells by a fluorescence-activated cell sorting system in afirst experiment of the present invention.

FIG. 7 shows the results of analyzing the effect of Lactobacillus brevisCH23, Bifidobacterium longum CH57 or a mixture thereof on ZO-1 proteinexpression in CaCO2 cells in a first experiment of the presentinvention.

FIG. 8 shows the colon appearance or myeloperoxidase (MPO) activityindicating the effect of Bifidobacterium longum CH57 on model animalshaving acute colitis induced by TNBS, in a first experiment of thepresent invention.

FIG. 9 depicts histological images showing the effect of Bifidobacteriumlongum CH57 on model animals having acute colitis induced by TNBS, in afirst experiment of the present invention.

FIG. 10 shows inflammation-related cytokine levels indicating the effectof Bifidobacterium longum CH57 on model animals having acute colitisinduced by TNBS, in a first experiment of the present invention.

FIG. 11 shows the colon appearance or myeloperoxidase (MPO) activityindicating the effect of Lactobacillus brevis CH23 on model animalshaving acute colitis induced by TNBS, in a first experiment of thepresent invention.

FIG. 12 depicts histological images of colon, which show the effect ofLactobacillus brevis CH23 on model animals having acute colitis inducedby TNBS, in a first experiment of the present invention.

FIG. 13 shows T-cell differentiation patterns indicating the effect ofLactobacillus brevis CH23 on model animals having acute colitis inducedby TNBS, in a first experiment of the present invention.

FIG. 14 shows inflammation-related cytokine levels indicating the effectof Lactobacillus brevis CH23 on model animals having acute colitisinduced by TNBS, in a first experiment of the present invention.

FIG. 15 shows the colon appearance or myeloperoxidase (MPO) activityindicating the effect of a mixture of Bifidobacterium longum CH57 andLactobacillus brevis CH23 on model animals having acute colitis inducedby TNBS, in a first experiment of the present invention.

FIG. 16 depicts histological images showing the effect of a mixture ofBifidobacterium longum CH57 and Lactobacillus brevis CH23 on modelanimals having acute colitis induced by TNBS, in a first experiment ofthe present invention.

FIG. 17 shows inflammation-related cytokine levels indicating the effectof a mixture of Bifidobacterium longum CH57 and Lactobacillus brevisCH23 on model animals having acute colitis induced by TNBS, in a firstexperiment of the present invention.

FIG. 18 shows weight changes indicating the effect of a mixture ofBifidobacterium longum CH57 and Lactobacillus brevis CH23 onobesity-induced model animals, in a first experiment of the presentinvention.

FIG. 19 shows the appearance of colon, myeloperoxidase (MPO) activity,histological images of colon, and the like indicating the effect of amixture of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 onobesity-induced model animals, in a first experiment of the presentinvention.

FIG. 20 shows inflammation-related cytokine levels indicating the effectof a mixture of Bifidobacterium longum CH57 and Lactobacillus brevisCH23 on obesity-induced model animals, in a first experiment of thepresent invention.

FIG. 21 shows inflammatory response markers indicating the effect of amixture of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 onobesity-induced model animals, in a first experiment of the presentinvention.

FIG. 22 shows the differentiation patterns of T cells into Th17 cellsindicating the effect of lactic acid bacteria on model animals havingacute colitis induced by TNBS, in a second experiment of the presentinvention.

FIG. 23 shows the differentiation patterns of T cells into Treg cellsindicating the effect of lactic acid bacteria on model animals havingacute colitis induced by TNBS, in a second experiment of the presentinvention.

FIG. 24 shows inflammatory response markers indicating the effect oflactic acid bacteria on model animals having acute colitis induced byTNBS, in a second experiment of the present invention.

FIG. 25 depicts images showing the effect of lactic acid bacteria on thestomach mucosa of mice having gastric ulcer induced by ethanol, in asecond experiment of the present invention.

FIG. 26 shows the gross gastric lesion score indicating the effect oflactic acid bacteria on the stomach mucosa of mice having gastric ulcerinduced by ethanol, in a second experiment of the present invention.

FIG. 27 shows the ulcer index indicating the effect of lactic acidbacteria on the stomach mucosa of mice having gastric ulcer induced byethanol, in a second experiment of the present invention.

FIG. 28 shows the histological activity index indicating the effect oflactic acid bacteria on the stomach mucosa of mice having gastric ulcerinduced by ethanol, in a second experiment of the present invention.

FIG. 29 shows the myeloperoxidase (MPO) activity indicating the effectof lactic acid bacteria on the stomach mucosa of mice having gastriculcer induced by ethanol, in a second experiment of the presentinvention.

FIG. 30 shows CXCL4 expression levels indicating the effect of lacticacid bacteria on the stomach mucosa of mice having gastric ulcer inducedby ethanol, in a second experiment of the present invention.

FIG. 31 shows TNF-α expression levels indicating the effect of lacticacid bacteria on the stomach mucosa of mice having gastric ulcer inducedby ethanol, in a second experiment of the present invention.

MODE FOR INVENTION

As used herein, terms used in the present invention will be defined.

As used herein, the term “culture” means a product obtained by culturinga microorganism in a known liquid medium or solid medium, and thus isintended to include a microorganism.

As used herein, the terms “pharmaceutically acceptable” and“sitologically acceptable” means neither significantly stimulating anorganism nor inhibiting the biological activity and characteristics ofan active material administered.

As used herein, the term “preventing” refers to all actions that inhibitsymptoms or delay the progression of a particular disease byadministrating the composition of the present invention.

As used herein, the term “treating” refers to all actions that alleviateor beneficially change the symptoms of a particular disease byadministering the composition of the present invention.

As used herein, the term “alleviating” refers to all actions that atleast reduce a parameter related to the condition to be treated, forexample, the degree of symptom.

As used herein, the term “administering” means providing the compositionof the present invention to a subject by any suitable method. As usedherein, the term “subject” means all animals, including humans, monkeys,dogs, goats, pigs or rats, which have a particular disease whosesymptoms can be alleviated by administering the composition of thepresent invention.

As used herein, the term “pharmaceutically effective amount” refers toan amount sufficient to treat diseases, at a reasonable benefit/riskratio applicable to any medical treatment. The pharmaceuticallyeffective amount may be determined depending on factors including thekind of subject's disease, the severity of the disease, the activity ofthe drug, sensitivity to the drug, the time of administration, the routeof administration, excretion rate, the duration of treatment and drugsused in combination with the composition, and other factors known in themedical field.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention is related to novel lactic acidbacteria having various physiological activities or to novel lactic acidbacteria mixture which may have increased physiological activities.

A novel lactic acid bacteria according to one embodiment of the presentinvention is selected from Lactobacillus brevis comprising a 16S rDNAnucleotide sequence represented by SEQ ID NO: 1, Bifidobacterium longumcomprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 3,Lactobacillus plantarum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 4, Lactobacillus plantarum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 5 or Bifidobacteriumlongum comprising a 16S rDNA nucleotide sequence represented by SEQ IDNO: 7, and has antioxidant activity, β-glucuronidase inhibitoryactivity, lipopolysaccharide (LPS) production inhibitory activity ortight junction protein expression-inducing activity.

The Lactobacillus brevis comprising the 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 1 is an anaerobic bacillus isolated fromkimchi, is positive to gram staining, can survive in a wide temperaturerange, low pHs and high salt concentrations, and produces glucosidase.Furthermore, the Lactobacillus brevis comprising the 16S rDNA nucleotidesequence represented by SEQ ID NO: 1 utilizes D-ribose, D-xylose,D-glucose, D-fructose, esculin, salicin, maltose, melibiose,5-keto-gluconate and the like as carbon sources. In addition, theLactobacillus brevis comprising the 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 1 is preferably Lactobacillus brevis CH23(accession number: KCCM 11762P). The Bifidobacterium longum comprisingthe 16S rDNA nucleotide sequence represented by SEQ ID NO: 3 is ananaerobic bacillus isolated from human feces, is positive to gramstaining, and produces glucosidase. Furthermore, the Bifidobacteriumlongum comprising the 16S rDNA nucleotide sequence represented by SEQ IDNO: 3 utilizes D-galactose, D-glucose, D-fructose and the like as carbonsources. In addition, the Bifidobacterium longum comprising the 16S rDNAnucleotide sequence represented by SEQ ID NO: 3 is preferablyBifidobacterium longum CH57 (accession number: KCCM 11764P). TheLactobacillus plantarum comprising the 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 4 is an anaerobic bacillus isolated fromkimchi and is positive to gram staining. Furthermore, the Lactobacillusplantarum comprising the 16S rDNA nucleotide sequence represented by SEQID NO: 4 utilizes D-ribose, D-galactose, D-glucose, D-fructose,D-mannose, mannitol, sorbitol, N-acetyl-glucosamine, amygdalin, arbutin,esculin, salicin, cellobiose, maltose, melibiose, sucrose, trehalose,melezitose and the like as carbon sources. In addition, theLactobacillus plantarum comprising the 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 4 is preferably Lactobacillus plantarum LC5(accession number: KCCM 11800P). The Lactobacillus plantarum comprisingthe 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 is ananaerobic bacillus isolated from kimchi, and is positive to gramstaining. Furthermore, the Lactobacillus plantarum comprising the 16SrDNA nucleotide sequence represented by SEQ ID NO: 5 utilizesL-arabinose, D-ribose, D-glucose, D-fructose, D-mannose, mannitol,sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin,cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitoseand the like as carbon sources. In addition, the Lactobacillus plantarumcomprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 5is preferably Lactobacillus plantarum LC27 (accession number: KCCM11801P). The Bifidobacterium longum comprising the 16S rDNA nucleotidesequence represented by SEQ ID NO: 7 is an anaerobic bacillus isolatedfrom human feces, and is positive to gram staining. Furthermore, theBifidobacterium longum comprising the 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 7 utilizes L-arabinose, D-xylose, D-glucose,D-fructose, esculin, maltose, lactose, melibiose, sucrose and the likeas carbon sources. In addition, the Bifidobacterium longum comprisingthe 16S rDNA nucleotide sequence represented by SEQ ID NO: 7 ispreferably Bifidobacterium longum LC67 (accession number: KCCM 11802P).

A mixture of lactic acid bacteria according to an embodiment of thepresent invention is a mixture of two or more lactic acid bacteriaselected from Lactobacillus brevis comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 1, Bifidobacterium longum comprisinga 16S rDNA nucleotide sequence represented by SEQ ID NO: 3,Lactobacillus plantarum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 4, Lactobacillus plantarum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 5 and Bifidobacteriumlongum comprising a 16S rDNA nucleotide sequence represented by SEQ IDNO: 7. In view of the synergistic effect of lactic acid bacteria, themixture of lactic acid bacteria according to the embodiment of thepresent invention is preferably a combination of Lactobacillus breviscomprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 1and Bifidobacterium longum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 3. In addition, in view of the synergisticeffect of lactic acid bacteria, the mixture of lactic acid bacteriaaccording to the embodiment of the present invention is preferably acombination of one or more Lactobacillus sp. selected from Lactobacillusplantarum comprising a 16S rDNA nucleotide sequence represented by SEQID NO: 4 or Lactobacillus plantarum comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 5; and Bifidobacterium longumcomprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 7.The mixture of lactic acid bacteria has higher antioxidant activity,β-glucuronidase inhibitory activity, lipopolysaccharide (LPS) productioninhibitory activity or tight junction protein expression-inducingactivity than a single lactic acid bacteria due to the synergisticeffect of a specific Lactobacillus sp. strain and a specificBifidobacterium sp. strain, and is more advantageous in terms offunctional food and medicinal materials. In the mixture of lactic acidbacteria according to the embodiment of the present invention, theLactobacillus brevis comprising the 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 1 is preferably Lactobacillus brevis CH23(accession number: KCCM 11762P); the Bifidobacterium longum comprisingthe 16S rDNA nucleotide sequence represented by SEQ ID NO: 3 ispreferably Bifidobacterium longum CH57 (accession number: KCCM 11764P);the Lactobacillus plantarum comprising the 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 4 is preferably Lactobacillus plantarum LC5(accession number: KCCM 11800P); the Lactobacillus plantarum comprisingthe 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 ispreferably Lactobacillus plantarum LC27 (accession number: KCCM 11801P);and the Bifidobacterium longum comprising the 16S rDNA nucleotidesequence represented by SEQ ID NO: 7 is preferably Bifidobacteriumlongum LC67 (accession number: KCCM 11802P).

Another aspect of the present invention is related to various uses ofthe novel lactic acid bacteria or the novel lactic acid bacteriamixture. As the use of the novel lactic acid bacteria, the presentinvention provides a composition for preventing, alleviating or treatingintestinal damage, liver injury, allergic disease, inflammatory diseaseor obesity comprising a lactic acid bacteria from Lactobacillus breviscomprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 1,Bifidobacterium longum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 3, Lactobacillus plantarum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 4, Lactobacillusplantarum comprising a 16S rDNA nucleotide sequence represented by SEQID NO: 5 or Bifidobacterium longum comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 7, a culture thereof, a lysatethereof or an extract thereof as an active ingredient. Furthermore, asthe use of the novel lactic acid bacteria mixture, the present inventionprovides a composition for preventing, alleviating or treatingintestinal damage, liver injury, allergic disease, inflammatory diseaseor obesity comprising a mixture of two or more lactic acid bacteriaselected from Lactobacillus brevis comprising a 16S rDNA nucleotidesequence represented by SEQ ID NO: 1, Bifidobacterium longum comprisinga 16S rDNA nucleotide sequence represented by SEQ ID NO: 3,Lactobacillus plantarum comprising a 16S rDNA nucleotide sequencerepresented by SEQ ID NO: 4, Lactobacillus plantarum comprising a 16SrDNA nucleotide sequence represented by SEQ ID NO: 5 and Bifidobacteriumlongum comprising a 16S rDNA nucleotide sequence represented by SEQ IDNO: 7, a culture thereof, a lysate thereof or an extract thereof as anactive ingredient. In the composition of the present invention, thetechnical characteristics of the Lactobacillus brevis, Lactobacillusplantarum and Bifidobacterium longum are as described above, and thusthe description thereof is omitted. The intestinal damage refers to acondition in which the function of the intestines (particularly smallintestine or large intestine) is abnormal due to intestinal floradisturbance or the like. Preferably, the intestinal damage is intestinalpermeability syndrome. Furthermore, the liver injury refers to acondition in which the function of the liver is abnormal due to externalfactors or internal factors. Preferably, the liver injury is selectedfrom hepatitis, fatty liver or liver cirrhosis. Furthermore, thehepatitis includes all non-alcoholic hepatitis and alcoholic hepatitis.Moreover, the fatty liver includes all non-alcoholic fatty liver andalcoholic fatty liver. Furthermore, the allergic disease is not limitedin its kind if it caused by excessive immune responses of a living body,and is preferably selected from atopic dermatitis, asthma, pharyngitisor chronic dermatitis. Furthermore, the inflammatory disease is notlimited in its kind if it caused by inflammatory responses, and ispreferably selected from gastritis, gastric ulcer, arthritis or colitis.Moreover, the arthritis includes rheumatoid arthritis. The colitisrefers to a condition in which inflammation occurred in the largeintestine due to bacterial infection or pathological fermentation ofintestinal contents. The colitis includes infectious colitis andnon-infectious colitis. Specific examples of the colitis includeinflammatory bowel diseases, irritable bowel syndrome and the like.Furthermore, the inflammatory bowel diseases include ulcerative colitis,Crohn's disease and the like.

In the present invention, a culture of the lactic acid bacteria or aculture of the lactic acid bacteria mixture is a produced by culturing acertain strain or a mixture of strains in a medium. The medium may beselected from known liquid media or solid media, and may be, forexample, MRS liquid medium, MRS agar medium or BL agar medium.

In the present invention, the composition may be embodied as apharmaceutical composition, a food additive, a food composition(particularly, a functional food composition), a feed additive or thelike depending on the intended use or aspect. In addition, the contentof the lactic acid bacteria or the lactic acid bacteria mixture as anactive ingredient may also be adjusted within a wide range depending onthe specific type, intended use or aspect of the composition.

The content of the novel lactic acid bacteria, the novel lactic acidbacteria mixture, a culture thereof, a lysate thereof or an extractthereof as an active ingredient in the pharmaceutical compositionaccording to the present invention is not particularly limited. Forexample, the content may be 0.01 to 99 wt %, preferably 0.5 to 50 wt %,more preferably 1 to 30 wt %, based on the total weight of thecomposition. In addition, the pharmaceutical composition according tothe present invention may further contain, in addition to the activeingredient, additives such as pharmaceutically acceptable carriers,excipients or diluents. Carriers, excipients and diluents, which may becontained in the pharmaceutical composition according to the presentinvention, include lactose, dextrose, sucrose, sorbitol, mannitol,xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin,calcium phosphate, calcium silicate, cellulose, methyl cellulose,microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate andmineral oil. In addition, the pharmaceutical composition according tothe present invention may further contain, in addition to the novellactic acid bacteria, the novel lactic acid bacteria mixture, a culturethereof, a lysate thereof or an extract thereof, one or more activeingredients having the effect of preventing or treating intestinaldamage, liver injury, allergic disease, inflammatory disease or obesity.The pharmaceutical composition according to the present invention may beprepared as formulations for oral administration or formulations forparenteral administration, and the formulations may be prepared usingdiluents or excipients, such as fillers, extenders, binders, wettingagents, disintegrants, surfactants and the like, which are commonlyused. Solid formulations for oral administration include tablets,pellets, powders, granules, capsules and the like, and such solidformulations may be prepared by mixing the active ingredient with atleast one excipient, for example, starch, calcium carbonate, sucrose,lactose or gelatin. In addition to simple excipients, lubricants such asmagnesium stearate or talc may also be used. Liquid formulations fororal administration include suspensions, solutions, emulsions and syrup,and may contain various excipients, for example, wetting agents,flavoring agents, aromatics, preservatives and the like, in addition towater and liquid paraffin which are frequently used simple diluents.Formulations for parenteral administration include sterilized aqueoussolutions, non-aqueous solutions, suspensions, emulsions, freeze-driedpreparations and suppositories. Propylene glycol, polyethylene glycol,plant oils such as olive oil, injectable esters such as ethyl oleate andthe like may be used as non-aqueous solvents or suspending agents. Asthe base of the suppositories, witepsol, Macrogol, Tween 61, cacaobutter, laurin fat, glycerogelatin and the like may be used.Furthermore, the composition may preferably be formulated depending oneach disease or component by a suitable method known in the art or themethod disclosed in Remington's Pharmaceutical Science (the latestedition), Mack Publishing Company, Easton Pa. The pharmaceuticalcomposition of the present invention may be administered orally orparenterally to mammals, including humans, according to a desiredmethod. Routes for parenteral administration include skin externalapplication, intraperitoneal injection, intrarectal injection,subcutaneous injection, intravenous injection, intramuscular injection,intrathoracic injection or the like. The dose of the pharmaceuticalcomposition of the present invention is not particularly limited as longas it is a pharmaceutically effective amount. The dose may varydepending on the patient's weight, age, sex, health condition, diet,administration time, administration mode, excretion rate and theseverity of the disease. The daily dose of the pharmaceuticalcomposition of the present invention is not particularly limited, but ispreferably 0.1 to 3000 mg/kg based on an active ingredient, morepreferably 1 to 2000 mg/kg based on an active ingredient and may beadministered once or several times a day.

Furthermore, the content of the novel lactic acid bacteria, the novellactic acid bacteria mixture, a culture thereof, a lysate thereof or anextract thereof as an active ingredient in the food compositionaccording to the present invention is 0.01 to 99 wt %, preferably 0.1 to50 wt %, more preferably 0.5 to 25 wt %, based on the total weight ofthe composition, but is not limited thereto. The food composition of thepresent invention may be in the form of pellets, powders, granules,infusions, tablets, capsules, liquid or the like, and specific examplesof the food may include meats, sausages, breads, chocolates, candies,snacks, confectioneries, pizzas, ramens, other noodles, gums, dairyproducts including ice creams, various kinds of soups, beverages, teas,functional water, drinks, alcoholic beverages, vitamin complexes and thelike, and may include all health foods in a general sense. The foodcomposition of the present invention may further contain sitologicallyacceptable carriers, various flavoring agents or natural carbohydratesas additional ingredients, in addition to the active ingredient.Additionally, the food composition of the present invention may containvarious nutrients, vitamins, electrolytes, flavoring agents, coloringagents, pectic acid and its salt, alginic acid and its salt, an organicacid, a protective colloidal thickener, a pH adjusting agent, astabilizer, a preservative, glycerin, alcohol, a carbonating agent usedfor carbonated drinks and the like. Additionally, the food compositionof the present invention may contain fruit flesh for preparing naturalfruit juices, fruit juice drinks and vegetable drinks. These ingredientsmay be used independently or as a mixture. The above-described naturalcarbohydrates may include monosaccharides such as glucose and fructose,disaccharides such as maltose and sucrose, polysaccharides such asdextrin and cyclodextrin and sugar alcohols such as xylitol, sorbitol,and erythritol. As a flavoring agent, a natural flavoring agent such asthaumatin or a stevia extract, or a synthetic flavoring agent such assaccharin or aspartame may be used.

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are merely intended to clearly illustrate the technicalcharacteristics of the present invention and do not limit the scope ofthe present invention.

I. First Experiment for Screening of Lactic Acid Bacteria and Evaluationof the Effects Thereof

1. Isolation and Identification of Lactic Acid Bacteria

(1) Isolation of Lactic Acid Bacteria from Kimchi

Each of Chinese cabbage kimchi, radish kimchi and green onion kimchi wascrushed, and the crushed liquid was suspended in MRS liquid medium (MRSBroth; Difco, USA). Next, the supernatant was collected, transferred toMRS agar medium (Difco, USA) and cultured anaerobically at 37° C. forabout 48 hours, and then strains that formed colonies were isolated.

(2) Isolation of Lactic Acid Bacteria from Human Feces

Human feces were suspended in GAM liquid medium (GAM broth; NissuiPharmaceutical, Japan). Next, the supernatant was collected, transferredto BL agar medium (Nissui Pharmaceutical, Japan) and culturedanaerobically at 37° C. for about 48 hours, and then Bifidobacterium sp.strains that formed colonies were isolated.

(3) Identification of Screened Lactic Acid Bacteria

The physiological characteristics and 16S rDNA sequences of the strainsisolated from kimchi or human feces were analyzed to identify thespecies of the strains, and names were given to the strains. Table 1below the control numbers and strain names of the lactic acid bacteriaisolated from Chinese cabbage kimchi, radish kimchi, green onion kimchiand human feces.

TABLE 1 Control No. Strain name 1 Lactobacillus acidophilus CH1 2Lactobacillus acidophilus CH2 3 Lactobacillus acidophilus CH3 4Lactobacillus brevis CH4 5 Lactobacillus curvatus CH5 6 Lactobacillusbrevis CH6 7 Lactobacillus casei CH7 8 Lactobacillus planantrum CH8 9Lactobacillus sakei CH9 10 Lactobacillus curvatus CH10 11 Lactobacillussakei CH11 12 Lactobacillus curvatus CH12 13 Lactobacillus plantarumCH13 14 Lactobacillus fermentum CH14 15 Lactobacillus fermentum CH15 16Lactobacillus gasseri CH16 17 Lactobacillus paracasei CH17 18Lactobacillus helveticus CH18 19 Lactobacillus helveticus CH19 20Lactobacillus johnsonii CH20 21 Lactobacillus johnsonii CH21 22Lactobacillus johnsonii CH22 23 Lactobacillus brevis CH23 24Lactobacillus paracasei CH24 25 Lactobacillus kimchi CH25 26Lactobacillus gasseri CH26 27 Lactobacillus paracasei CH27 28Lactobacillus pentosus CH28 29 Lactobacillus pentosus CH29 30Lactobacillus reuteri CH30 31 Lactobacillus sakei CH31 32 Lactobacillusjohnsonii CH32 33 Lactobacillus sakei CH33 34 Lactobacillus sakei CH3435 Lactobacillus plantarum CH35 36 Lactobacillus sanfranciscensis CH3637 Bifidobacterium pseudocatenulatum CH37 38 Bifidobacteriumpseudocatenulatum CH38 39 Bifidobacterium adolescentis CH39 40Bifidobacterium adolescentis CH40 41 Bifidobacterium adolescentis CH4142 Bifidobacterium animalis CH42 43 Bifidobacterium animalis CH43 44Bifidobacterium bifidum CH44 45 Bifidobacterium bifidum CH45 46Bifidobacterium breve CH46 47 Bifidobacterium breve CH47 48Bifidobacterium breve CH48 49 Bifidobacterium catenulatum CH49 50Bifidobacterium catenulatum CH50 51 Bifidobacterium dentium CH51 52Bifidobacterium infantis CH52 53 Bifidobacterium infantis CH53 54Bifidobacterium infantis CH54 55 Bifidobacterium longum CH55 56Bifidobacterium longum CH56 57 Bifidobacterium longum CH57 58Bifidobacterium longum CH58 59 Bifidobacterium longum CH59 60Bifidobacterium longum CH60

Among the strains shown in Table 1 above, Lactobacillus brevis CH23 wasa gram-positive anaerobic bacillus, did not form spores, and couldsurvive even under aerobic conditions. Furthermore, Lactobacillus brevisCH23 survived at 10 to 42° C. and was an acid-resistant strain stable atpH 2 for 2 hours. Furthermore, Lactobacillus brevis CH23 survived evenin 2% sodium chloride solution and actively produced glucosidase. Inaddition, to chemically classify Lactobacillus brevis CH23, the 16S rDNAthereof was analyzed, and as a result, it was shown that Lactobacillusbrevis CH23 had a nucleotide sequence of SEQ ID NO: 1. The 16S rDNAnucleotide sequence of Lactobacillus brevis CH23 was identified by BLASTin the Genebank (http://www.ncbi.nlm.nih.gov/), and as a result, aLactobacillus brevis strain having the same 16S rDNA nucleotide sequenceas that of Lactobacillus brevis CH23 was not found, and Lactobacillusbrevis CH23 showed a homology of 99% with the 16S rDNA sequence ofLactobacillus brevis strain FJ004.

Among the strains shown in Table 1 above, Lactobacillus johnsonii CH32was a gram-positive anaerobic bacillus, did not form spores, and couldsurvive under aerobic conditions. Furthermore, Lactobacillus johnsoniiCH32 survived stably at a temperature of up to 45° C., and was anacid-resistant strain stable in pH 2 for 2 hours. Moreover,Lactobacillus johnsonii CH32 actively produced β-glucosidase, but didnot produce β-glucuronidase. In addition, to chemically classifyLactobacillus johnsonii CH32, the 16S rDNA thereof was analyzed, and asa result, it was shown that Lactobacillus johnsonii CH32 had anucleotide sequence of SEQ ID NO: 2. The 16S rDNA nucleotide sequence ofLactobacillus johnsonii CH32 was identified by BLAST in Genebank(http://www.ncbi.nlm.nih.gov/), and as a result, a Lactobacillusjohnsonii strain having the same 16S rDNA nucleotide sequence as that ofLactobacillus johnsonii CH32 was not found, and Lactobacillus johnsoniiCH32 showed a homology of 99% with the 16S rDNA sequence ofLactobacillus johnsonii strain JCM 2012.

Among the strains shown in Table 1 above, Bifidobacterium longum CH57was a gram-positive anaerobic bacillus, did not form spores, and showedvery low viability under aerobic conditions. Furthermore,Bifidobacterium longum CH57 was thermally unstable. Furthermore,Bifidobacterium longum CH57 actively produced glucosidase, but did notproduce β-glucuronidase. In addition, to chemically classifyBifidobacterium longum CH57, the 16S rDNA thereof was analyzed, and as aresult, it was shown that Bifidobacterium longum CH57 had a nucleotidesequence of SEQ ID NO: 3. The 16S rDNA nucleotide sequence ofBifidobacterium longum CH57 was identified by BLAST in the Genebank(http://www.ncbi.nlm.nih.gov/), and as a result, a Bifidobacteriumlongum strain having the same 16S rDNA nucleotide sequence as that ofBifidobacterium longum CH57 was not found, and Bifidobacterium longumCH57 showed a homology of 99% with the 16S rDNA sequence ofBifidobacterium longum strain CBT-6.

In addition, among the physiological characteristics of Lactobacillusbrevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longumCH57, the carbon source utilization was analyzed using a sugarfermentation by an API kit (model: API 50 CHL; manufactured byBioMerieux's, USA). Table 2 below shows the results of analyzing thecarbon source utilization of Lactobacillus brevis CH23; Table 3 belowshows the results of analyzing the carbon source utilization ofLactobacillus johnsonii CH32; and Table 4 below shows the results ofanalyzing the carbon source utilization of Bifidobacterium longum CH57.In Tables 2, 3 and 4, “+” indicates the case in which carbon sourceutilization is positive; “−” indicates the case in which carbon sourceutilization is negative; and “±” indicates the case in which carbonsource utilization is ambiguous. As shown in Tables 2, 3 and 4 below,Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 andBifidobacterium longum CH57 showed carbon source utilization differentfrom that of other strains of the same species with respect to somecarbon sources.

TABLE 2 Strain name Strain name L. brevis L. brevis Carbon source L.brevis ¹⁾ CH23 Carbon source L. brevis ¹⁾ CH23 glycerol − − salicin + +erythritol − − cellobiose + − D-arabinose − − maltose + + L-arabinose +− lactose + − D-ribose + + melibiose − + D-xylose + + sucrose + −L-xylose − − trehalose + − D-adonitol − − inulin + −methyl-β-D-xylopyranoside − − melezitose + − D-galactose + − raffinose −− D-glucose + + starch − − D-fructose + + glycogen − − D-mannose + −xylitol − − L-sorbose − − gentiobiose + − L-rhamnose − − D-turanose + −dulcitol + − D-lyxose − − inositol − − D-tagatose + − mannitol + −D-fucose − − sorbitol + − L-fucose − − α-methyl-D-mannoside − −D-arabitol − − α-methly-D-glucoside − − L-arabitol − −N-acetyl-glucosamine + ± gluconate + ± amygdalin + − 2-keto-gluconate −− arbutin + − 5-keto-gluconate − + esculin + + ¹⁾Suriasih K., Aryanta WR, MahardikaG, Astawa N M. Microbiological and Chemical Properties ofKefir Made of Bali Cattle Milk. Food Science and Quality Management2012; 6: 112-22.

TABLE 3 Strain name Strain name L. johnsonii ²⁾ L. johnsonii ²⁾ Carbonsource L. johnsonii ²⁾ CH32 Carbon source L. johnsonii ²⁾ CH32 glycerol− − salicin − − erythritol − − cellobiose + − D-arabinose − − maltose− + L-arabinose − − lactose − + D-ribose − − melibiose + − D-xylose − −sucrose + + L-xylose − − trehalose + − D-adonitol − − inulin − −methyl-β-D-xylopyranoside − − melezitose − − D-galactose − − raffinose +− D-glucose − + starch − − D-fructose − + glycogen − − D-mannose + +xylitol − − L-sorbose − − gentiobiose − + L-rhamnose − − D-turanose − −dulcitol − − D-lyxose − − inositol − − D-tagatose − − mannitol − −D-fucose − − sorbitol − − L-fucose − − α-methyl-D-mannoside − −D-arabitol − − α-methly-D-glucoside − − L-arabitol − −N-acetyl-glucosamine + + gluconate − − amygdalin − − 2-keto-gluconate −− arbutin − − 5-keto-gluconate − − esculin − − ²⁾Pridmore R D, Berger B,Desiere F, Vilanova D, Barretto C, Pittet A C, Zwahlen M C, Rouvet M,Altermann E, Barrangou R, Mollet B, Mercenier A, Klaenhammer T, ArigoniF, Schell M A. The genome sequence of the probiotic intestinal bacteriumLactobacillus johnsonii NCC 533. Proc Natl Acad Sci USA. 2004 Feb. 24;101(8): 2512-7.

TABLE 4 Strain name Strain name B. longum ³⁾ B. longum ³⁾ Carbon sourceB. longum ³⁾ CH57 Carbon source B. longum ³⁾ CH57 glycerol ± − salicin ±− erythritol − − cellobiose ± ± D-arabinose − − maltose − − L-arabinose− − lactose − − D-ribose ± − melibiose − − D-xylose − − sucrose + ±L-xylose − − trehalose ± − D-adonitol − − inulin − −methyl-β-xylopyranoside − − melezitose − − D-galactose + + raffinose − −D-glucose + + starch − − D-fructose + + glycogen − − D-mannose − −xylitol − − L-sorbose − − gentiobiose − − L-rhamnose − − D-turanose − −dulcitol − − D-lyxose − − inositol − − D-tagatose − − mannitol + −D-fucose − − sorbitol − − L-fucose − − α-methyl-D-mannoside − −D-arabitol − − α-methly-D-glucoside − − L-arabitol − −N-acetyl-glucosamine ± − gluconate ± − amygdalin − − 2-keto-gluconate −− arbutin ± − 5-keto-gluconate − − esculin − − ³⁾Lukacova D, KarovucovaJ, Greifova M, Greif G, Sovcikova A, Kohhajdova Z. In vitro testing ofselected probiotic characteristics of Lactobacillus plantarum andBifidobacterium longum. Journal of Food and Nutrition Research 2006; 45:77-83.

(4) Information on Deposition of Lactic Acid Bacteria

The present inventors deposited Lactobacillus brevis CH23 with theKorean Culture Center of Microorganisms (address: Yurim Building, 45,Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an internationaldepositary authority, on Sep. 1, 2015 under accession number KCCM11762P. Furthermore, the present inventors deposited Lactobacillusjohnsonii CH32 with the Korean Culture Center of Microorganisms(address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul,Korea), an international depositary authority, on Sep. 1, 2015, underaccession number KCCM 11763P. Furthermore, the present inventorsdeposited Bifidobacterium longum CH57 with the Korean Culture Center ofMicroorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil,Seodaemun-gu, Seoul, Korea), an international depositary authority, onSep. 1, 2015 under accession number KCCM 11764P.

2. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation ofIntestinal Damage or Intestinal Permeability

In order to evaluate the effect of the lactic acid bacteria isolatedfrom kimchi or human feces, on the alleviation of intestinal damage orinternal permeability, the antioxidant activity, lipopolysaccharide(LPS) production inhibitory activity, β-glucuronidase (harmfulintestinal enzyme) inhibitory activity and tight junction proteinexpression-inducing activity of the lactic acid bacteria were measured.

(1) Experimental Methods

*Antioxidant Activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in ethanol to aconcentration of 0.2 mM to prepare a DPPH solution. A lactic acidbacteria suspension (1×10⁸ CFU/ml) or a vitamin C solution (1 g/ml) wasadded to 0.1 ml of the DPPH solution and cultured at 37° C. for 20minutes. The culture was centrifuged at 3000 rpm for 5 minutes, and thesupernatant was collected. Next the absorbance of the supernatant at 517nm was measured, and the antioxidant activity of the lactic acidbacteria was calculated.

*Lipopolysaccharide (LPS) Production-Inhibitory Activity

0.1 g of human fresh feces was suspended in 0.9 ml of sterilephysiological saline and diluted 100-fold with general anaerobic mediumto prepare a fecal suspension. 0.1 ml of the fecal suspension and 0.1 mlof lactic acid bacteria (1×10⁴ or 1×10⁵ CFU) were added to 9.8 ml ofsterile anaerobic medium (Nissui Pharmaceuticals, Japan) and culturedanaerobically for 24 hours. Next, the culture was sonicated for about 1hour to disrupt the outer cell membrane of the bacteria, and centrifugedat 5000×g, and the supernatant was collected. Next, the content of LPS(lipopolysaccharide) (which is a typical endotoxin) in the supernatantwas measured by a LAL (Limulus Amoebocyte Lysate) assay kit(manufactured by Cape Cod Inc., USA). In addition, in order to evaluatethe E. coli proliferation inhibitory activity of the lactic acidbacteria, the culture obtained through the same experiment as describedabove was diluted 1000-fold and 100000-fold and cultured in DHL medium,and then the number of E. coli cells was counted.

*β-Glucuronidase Inhibitory Activity

0.1 ml of 0.1 mM p-nitrophenyl-β-D-glucuronide solution, 0.2 ml of 50 mMphosphate buffered saline and 0.1 ml of a lactic acid bacteriasuspension (prepared by suspending of a lactic acid bacteria culture in5 ml of physiological saline) were placed in a reactor and subjected toan β-glucuronidase enzymatic reaction, and 0.5 ml of 0.1 mM NaOHsolution was added to stop the reaction. Next, the reaction solution wascentrifuged at 3000 rpm for 5 minutes, and the supernatant wascollected. Then, the absorbance of the supernatant at 405 nm wasmeasured.

*Tight Junction Protein Expression-Inducing Activity

Caco2 cells obtained from the Korean Cell Line Bank were cultured inRPMI 1640 medium for 48 hours, and then the cultured Caco2 cells weredispensed to each well of a 12-well plate at a density of 2×10⁶cells/well. Next, each well was treated with 1 μg of LPS(lipopolysaccharide) or a combination of 1 μg of LPS(lipopolysaccharide) and 1×10³ CFU of lactic acid bacteria and incubatedfor 24 hours. Next, the cultured cells were collected from each well,and the expression level of tight junction protein ZO-1 in the cells wasmeasured by an immunoblotting method.

(2) Experimental Results

The antioxidant activity, lipopolysaccharide (LPS) production inhibitoryactivity, β-glucuronidase inhibitory activity and tight junction proteinexpression-inducing activity of the lactic acid bacteria isolated fromkimchi or human feces were measured, and the results of the measurementare shown in Tables 5 and 6 below. As shown in Tables 5 and 6 below,Lactobacillus curvatus CH5, Lactobacillus sakei CH11, Lactobacillusbrevis CH23, Lactobacillus johnsonii CH32, Bifidobacteriumpseudocatenulatum CH38 and Bifidobacterium longum CH57 had excellentantioxidant activity, strongly inhibited lipopolysaccharide (LPS)production and β-glucuronidase activity, and strongly induced theexpression of tight junction protein. These lactic acid bacteria have anexcellent antioxidant effect, have an excellent effect of inhibiting theenzymatic activity of intestinal flora's harmful bacteria associatedwith inflammation and carcinogenesis, inhibit the production ofendotoxin LPS (lipopolysaccharide) produced by intestinal flora'sharmful bacteria, and induce the expression of tight junction protein.Thus, these lactic acid bacteria can improve intestinal permeabilitysyndrome.

TABLE 5 Tight junction protein Beta- LPS production expression ControlAntioxidant glucuronidase inhibitory inducing No. Strain name activityinhibitory activity activity activity 1 Lactobacillus acidophilusCH1 + + − − 2 Lactobacillus acidophilus CH2 + + + − 3 Lactobacillusacidophilus CH3 + + + − 4 Lactobacillus brevis CH4 + + − − 5Lactobacillus curvatus CH5 +++ + +++ ++ 6 Lactobacillus brevis CH6 + + −− 7 Lactobacillus casei CH7 + + − − 8 Lactobacillus planantrum CH8 + + −− 9 Lactobacillus sakei CH9 − + − − 10 Lactobacillus curvatus CH10 − + −− 11 Lactobacillus sakei CH11 +++ + +++ ++ 12 Lactobacillus curvatusCH12 − + − + 13 Lactobacillus plantarum CH13 − + − − 14 Lactobacillusfermentum CH14 − + − − 15 Lactobacillus fermentum CH15 +++ + ++ − 16Lactobacillus gasseri CH16 + + − − 17 Lactobacillus paracasei CH17 + + −− 18 Lactobacillus helveticus CH18 + + − − 19 Lactobacillus helveticusCH19 + + − − 20 Lactobacillus johnsonii CH20 + + − + 21 Lactobacillusjohnsonii CH21 + + − + 22 Lactobacillus johnsonii CH22 + + − + 23Lactobacillus brevis CH23 +++ + ++ ++ 24 Lactobacillus paracaseiCH24 + + − − 25 Lactobacillus kimchi CH25 + + − − 26 Lactobacillusgasseri CH26 + + − − 27 Lactobacillus paracasei CH27 + + − + 28Lactobacillus pentosus CH28 + + − − 29 Lactobacillus pentosus CH29 + + −− 30 Lactobacillus reuteri CH30 + − − −

TABLE 6 Tight junction protein Beta- LPS production expression ControlAntioxidant glucuronidase inhibitory inducing No. Strain name activityinhibitory activity activity activity 31 Lactobacillus sakei CH31 − +− + 32 Lactobacillus johnsonii CH32 +++ + ++ ++ 33 Lactobacillus sakeiCH33 + + − + 34 Lactobacillus sakei CH34 + + − + 35 Lactobacillusplantanrum CH35 + + + + 36 Lactobacillus sanfranciscensis CH36 + + + +37 Bifidobacterium pseudocatenulatum CH37 − + − + 38 Bifidobacteriumpseudocatenulatum CH38 +++ + ++ ++ 39 Bifidobacterium adolescentis CH39− + − + 40 Bifidobacterium adolescentis CH40 − + +++ + 41Bifidobacterium adolescentis CH41 + + − + 42 Bifidobacterium animalisCH42 + + − − 43 Bifidobacterium animalis CH43 + + − − 44 Bifidobacteriumbifidum CH44 + + − − 45 Bifidobacterium bifidum CH45 + + − − 46Bifidobacterium breve CH46 + − − − 47 Bifidobacterium breve CH47 + + − +48 Bifidobacterium breve CH48 + + − + 49 Bifidobacterium catenulatumCH49 + + − ++ 50 Bifidobacterium catenulatum CH50 − + − − 51Bifidobacterium dentium CH51 + − − − 52 Bifidobacterium infantis CH52− + − − 53 Bifidobacterium infantis CH53 − + − − 54 Bifidobacteriuminfantis CH54 + + − − 55 Bifidobacterium longum CH55 + + − + 56Bifidobacterium longum CH56 +++ + ++ + 57 Bifidobacterium longum CH57+++ + +++ ++ 58 Bifidobacterium longum CH58 + + + + 59 Bifidobacteriumlongum CH59 + + + + 60 Bifidobacterium longum CH60 + − + + * The finalconcentration of lactic acid bacteria in measurement of antioxidantactivity: 1 × 10⁴ CFU/ml; the concentration of lactic acid bacteriaadded for measurement of beta-glucuronidase inhibitory activity andlipopolysaccharide (LPS) production inhibitory activity: 1 × 10⁴ CFU/ml;the concentration of lactic acid bacteria in measurement of tightjunction protein expression-inducing activity: 1 × 10⁴ CFU/ml. *Criteria for measurement of various activities of lactic acid bacteria:very strongly (+++; >90%); strongly (++; >60-90%); weakly (+; >20-60%);not or less than 20% (−; <20%).

3. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation ofLiver Injury

Based on evaluation of the effect of the lactic acid bacteria on thealleviation of intestinal damage or intestinal permeability syndrome,the following seven strains were selected: Lactobacillus curvatus CH5,Lactobacillus sakei CH11, Lactobacillus fermentum CH15, Lactobacillusbrevis CH23, Lactobacillus johnsonii CH32, Bifidobacteriumpseudocatenulatum CH38 and Bifidobacterium longum CH57. The effect ofeach of these selected strains or a mixture of these strains on thealleviation of liver injury was evaluated using various liver injurymodel animals.

(1) Measurement of the Liver Injury-Alleviating Effect of Lactic AcidBacteria by an Experiment Using Model Animals Having Liver InjuryInduced by D-Galactosamine

1) Experimental Method

Mice (C57BL/6, male) were divided into several groups, each consistingof 6 animals. D-Galactosamine was administered intraperitoneally to thetest animals of groups other than a normal group at a dose of 800 mg/kgto induce liver injury. From 2 hours after intraperitonealadministration of D-galactosamine, 1×10⁹ CFU of lactic acid bacteriawere administered orally to the test animals of groups other than thenormal group and the negative control group, once a day for 3 days. Inaddition, silymarin in place of lactic acid bacteria was administeredorally to the test animals of the positive control group at a dose of100 mg/kg, once a day for 3 days. At 6 hours after the lastadministration of the drug, blood was taken from the heart. The takenblood was allowed to stand at room temperature for 60 minutes, andcentrifuged at 3,000 rpm for 15 minutes to separate serum. The GPT(glutamic pyruvate transaminase) and GOT (glutamic oxalacetictransaminase) levels in the separated serum were measured using a bloodassay kit (ALT & AST measurement kit; Asan Pharm. Co., Korea).

In addition, liver tissue was dissected from the test animals, and theamount of malondialdehyde (MDA) present in the liver tissue wasmeasured. Malondialdehyde is a marker of lipid peroxidation.Specifically, 0.5 g of the dissected liver tissue was added to a 16-foldvolume of RIPA solution (0.21M mannitol, 0.1M EDTA-2Na, 0.07M sucrose,0.01M Trizma base), and then homogenized using a homogenizer. Thehomogenized solution was centrifuged at 3,000 rpm for 10 minutes, andthe liver homogenate was collected. 0.5 ml of the liver homogenate wasadded to 0.4 ml of 10% SDS, incubated at 37° C. for 30 minutes, andcooled, and then 3 ml of 1% phosphate buffer and 1 ml of 0.6% TBA wereadded thereto and heated on a water bath at 100° C. for 45 minutes todevelop the sample solution. The developed sample solution was added toand mixed with 4 ml of n-butanol, and then centrifuged at 3000 rpm for10 minutes, and the supernatant was collected. The absorbance of thecollected supernatant at 535 nm was measured to quantify MDA. Inaddition, a calibration curve for MDA measurement was plotted using1,1,3,3-tetraethoxypropane.

2) Experimental Results

FIG. 1 is a graph showing the change in GOT value when lactic acidbacteria were administered to model animals having liver injury inducedby D-galactosamine; FIG. 2 is a graph showing the change in GPT valuewhen lactic acid bacteria were administered to model animals havingliver injury induced by D-galactosamine; and FIG. 3 is a graph showingthe change in MDA value when lactic acid bacteria were administered tomodel animals having liver injury induced by D-galactosamine.

On the x-axis of FIGS. 1 to 3, “Nor” indicates a normal group; “Con”indicates a negative control group in which any drugs were notadministered to model animals having liver injury induced byD-galactosamine; “ch11” indicates a group administered withLactobacillus sakei CH11; “ch15” indicates a group administered withLactobacillus fermentum CH15; “ch23” indicates a group administered withLactobacillus brevis CH23; “ch32” indicates a group administered withLactobacillus johnsonii CH32; “ch38” indicates a group administered withBifidobacterium pseudocatenulatum CH38; “ch57” indicates a groupadministered with Bifidobacterium longum CH57; “ch57+ch11” indicates agroup administered with a lactic acid bacteria mixture prepared bymixing Bifidobacterium longum CH57 and Lactobacillus sakei CH11 in thesame amount; “ch57+ch23” indicates a group administered with a lacticacid bacteria mixture prepared by mixing Bifidobacterium longum CH57 andLactobacillus brevis CH23 in the same amount; “ch57+ch32” indicates agroup administered with a lactic acid bacteria mixture prepared bymixing Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32 inthe same amount; and “SM” indicates a positive control groupadministered with silymarin instead of lactic acid bacteria.

As shown in FIGS. 1 to 3, when each of Lactobacillus brevis CH23,Lactobacillus johnsonii CH32 and Bifidobacterium longum CH57 wasadministered to the model animals in which GOT, GPT and MAD valuesincreased due to liver injury, the liver injury was alleviated.Particularly, when a lactic acid bacteria mixture of Bifidobacteriumlongum CH57 and Lactobacillus brevis CH23 or a lactic acid bacteriamixture of Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32was administered, the liver injury was greatly alleviated. In addition,specific lactic acid bacteria or a mixture of lactic acid bacteriaselected therefrom showed a better effect on the alleviation of liverinjury than silymarin which is used as a drug for treating liver injury.These results suggest that specific lactic acid bacteria or a mixture oflactic acid bacteria selected therefrom is effective in alleviatingfatty liver induced by alcohol and high-fat diets, or in alleviatingliver diseases resulting from oxidative stress.

(2) Measurement of the Liver Injury-Alleviating Effect of Lactic AcidBacteria by an Experiment Using Model Animals Having Liver InjuryInduced by Tert-Butylperoxide

1) Experimental Method

Mice (C57BL/6, male) were divided into several groups, each consistingof 6 animals. Tert-butylperoxide was administered intraperitoneally tothe test animals of groups other than a normal group in an amount of 2.5mmol/kg to induce liver injury. From 2 hours after intraperitonealadministration of tert-butylperoxide, 2×10⁹ CFU of lactic acid bacteriawere administered orally to the test animals of groups other than thenormal group and the negative control group, once a day for 3 days. Inaddition, silymarin in place of lactic acid bacteria was administeredorally to the test animals of the positive control group at a dose of100 mg/kg, once a day for 3 days. At 6 hours after the lastadministration of the drug, blood was taken from the heart. The takenblood was allowed to stand at room temperature for 60 minutes, andcentrifuged at 3,000 rpm for 15 minutes to separate serum. The GPT(glutamic pyruvate transaminase) and GOT (glutamic oxalacetictransaminase) levels in the separated serum were measured using a bloodassay kit ((ALT & AST measurement kit; Asan Pharm. Co., Korea).

2) Experimental Results

Table 7 below shows the changes in GOT and GPT values when lactic acidbacteria were administered to the model animals having liver injuryinduced by tert-butylperoxide. As shown in Table 7 below, Lactobacillusbrevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longumCH57 showed excellent effects on the alleviation of liver injurycompared to silymarin, and a lactic acid bacteria mixture ofBifidobacterium longum CH57 and Lactobacillus brevis CH23 or a lacticacid bacteria mixture of Bifidobacterium longum CH57 and Lactobacillusjohnsonii CH32 showed a better effect on the alleviation of liverinjury.

TABLE 7 GOT GPT Test groups (IU/L) (IU/L) Normal group 36.1 26.3Negative control group 84.1 96.1 Group administered with CH23 58.0 74.2Group administered with CH32 53.0 70.5 Group administered with CH57 57.671.2 Group administered with CH57 + CH23 48.6 64.3 Group administeredwith CH57 + CH32 51.2 68.4 Group administered with silymarin 61.7 69.1

In Table 7 above, “CH23” indicates Lactobacillus brevis CH23; “CH32”indicates Lactobacillus johnsonii CH32; “CH57” indicates Bifidobacteriumlongum CH57; “CH57+CH23” indicates a lactic acid bacteria mixtureprepared by mixing Bifidobacterium longum CH57 and Lactobacillus brevisCH23 in the same amount; and “CH57+CH32” indicates a lactic acidbacteria mixture prepared by mixing Bifidobacterium longum CH57 andLactobacillus johnsonii CH32 in the same amount.

4. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation ofAllergy

(1) Measurement of the Inhibition of Degranulation by Lactic AcidBacteria

The RBL-2H3 cell line (rat mast cell line, the Korean Cell Line Bank,Cat. No. 22256) was cultured with DMEM (Dulbeccos' modified Eagle'smedium, Sigma, 22256) containing 10% FBS (fetal bovine serum) andL-glutamine in a humidified 5% CO₂ incubator at 37° C. The cellscontained in the culture medium were floated using trypsin-EDTAsolution, and the floated cells were isolated, collected and used in theexperiment. The collected RBL-2H3 cells were dispensed into a 24-wellplate at a density of 5×10⁵ cells/well and sensitized by incubation with0.5 μg/ml of mouse monoclonal IgE for 12 hours. The sensitized cellswere washed with 0.5 ml of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4mM MgCl₂, 25 mM PIPES, 40 mM NaOH, pH 7.2), and then incubated with 0.16ml of siraganian buffer (supplemented with 5.6 mM glucose, 1 mM CaCl₂,0.1% BSA) at 37° C. for 10 minutes. Next, lactic acid bacteria as a testdrug were added to the cell culture to a concentration of 1×10⁴ CFU/ml,or 0.04 ml of DSCG (disodium cromoglycate) as a control drug was addedto the cell culture, and after 20 minutes, the cells were activated with0.02 ml of antigen (1 μg/ml DNP-BSA) at 37° C. for 10 minutes. Next, thecell culture was centrifuged at 2000 rpm for 10 minutes, and thesupernatant was collected. 0.025 ml of the collected supernatant wastransferred to a 96-well plate, and then 0.025 ml of 1 mM p-NAG (asolution of p-nitrophenyl-N-acetyl-β-D-glucosamide in 0.1M citratebuffer, pH 4.5) was added thereto, and then the mixture was allowed toreact at 37° C. for 60 minutes. Next, the reaction was stopped byaddition of 0.2 ml of 0.1M Na₂CO₃/NaHCO₃, and the absorbance at 405 nmwas measured by an ELISA analyzer.

(2) Measurement of the Inhibition of Itching by Lactic Acid Bacteria

BALB/c mice were divided into several groups, each consisting of 5animals. 1×10⁹ CFU of lactic acid bacteria as a test drug wereadministered orally to test groups other than a normal group and acontrol group, once a day for 3 days, or DSCG (disodium cromoglycate) orAzelastine as a control drug was administered orally in an amount of 0.2mg/mouse, once a day for 3 days. At 1 hour after the last administrationof the drug, the mice were allowed to stand in an observation cage (24cm×22 cm×24 cm) for 10 minutes so as to be acclimated to theenvironment, and then the back portion of the head was shaved. Next, themice of the normal group were injected with physiological saline, andthe mice of the other test groups were injected with an itching inducer(50 μg of compound 48/80; Sigma, USA) by a 29-gauge needle. Next, eachmouse was immediately confined in an observation cage, and the itchingbehavior was observed under the unattended condition by recording withan 8-mm video camera (SV-K80, Samsung) for 1 hour. Scratching theinjection area with the back foot was regarded as the itching behavior,and scratching other portions was not regarded.

(3) Measurement of the Inhibition of Vascular Permeability by LacticAcid Bacteria

It is known that itching-induced areas have increased vascularpermeability. This experiment was performed in order to examine whetherlactic acid bacteria could efficiently inhibit vascular permeabilitycaused by various compounds. According to the same method as theabove-described experiment for measurement of itching inhibitoryactivity, the drug was administered to the same mice. Next,physiological saline was injected subcutaneously into the back portionof the head of the mice of the normal group, and an itching inducer (50μg of compound 48/80; Sigma, USA) was injected subcutaneously into theback portion of the head of the mice of the other test group. Next, 0.2ml of 1% Evans blue solution (Sigma, USA) was injected into the tailvein, and after 1 hour, the mice were euthanized. The skin of thesubcutaneously injected area was dissected, and incubated in 1 ml of 1NKOH at 37° C. overnight. On the next day, the incubated skin tissue wasmixed with 4 ml of 0.6N phosphoric acid-acetone (5:13) mixture andcentrifuged at 3000 rpm for 15 minutes, and the supernatant wascollected and measured for absorbance at 620 nm. Inhibition (%) ofvascular permeability was calculated using the following equation:

Inhibition (%)={1−[absorbance of area treated with drug and itchinginducer−absorbance of area not treated with itching inducer]/[absorbanceof area treated with itching inducer−absorbance of area not treated withitching inducer]}×100

(4) Experimental Results

Table 8 below shows the results of measuring the degranulationinhibition rate, itching inhibition rate and capillary permeabilityinhibition rate of the lactic acid bacteria. In Table 8 below, “CH5”indicates Lactobacillus curvatus CH5; “CH11” indicates Lactobacillussakei CH11; “CH15” indicates Lactobacillus fermentum CH15; “CH23”indicates Lactobacillus brevis CH23; “CH32” indicates Lactobacillusjohnsonii CH32; “CH38” indicates Bifidobacterium pseudocatenulatum CH38;“CH57” indicates Bifidobacterium longum CH57; “CH57+CH11” indicates alactic acid bacteria mixture prepared by mixing Bifidobacterium longumCH57 and Lactobacillus sakei CH11 in the same amount; “CH57+CH23”indicates a lactic acid bacteria mixture prepared by mixingBifidobacterium longum CH57 and Lactobacillus brevis CH23 in the sameamount; and “CH57+CH32” indicates a lactic acid bacteria mixtureprepared by mixing Bifidobacterium longum CH57 and Lactobacillusjohnsonii CH32 in the same amount.

As shown in Table 8 below, Lactobacillus curvatus CH5, Lactobacillusbrevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longumCH57 effectively inhibited the degranulation of basophils, andBifidobacterium longum CH57 very strongly inhibited itching and vascularpermeability. In addition, in comparison with these lactic acid bacteriaalone, a mixture of these lactic acid bacteria, particularly a mixtureof Bifidobacterium longum CH57 and Lactobacillus brevis CH23 or amixture of Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32showed higher degranulation inhibition rate, itching inhibition rate andvascular permeability inhibition rate. Thus, it can be seen that theselactic acid bacteria or mixtures thereof can very effectively alleviateallergic atopy, asthma, pharyngitis, chronic dermatitis or the like.

TABLE 8 Inhibition (%) vascular Drug Degranulation Itching permeabilityNone 0 2 1 CH5 53 46 45 CH11 47 46 45 CH15 48 42 42 CH23 54 47 47 CH3252 45 46 CH38 44 45 42 CH57 55 55 52 CH57 + CH11 59 56 54 CH57 + CH23 6362 61 CH57 + CH32 61 58 56 DSCG (disodium cromoglycate) 62 25 37Azelastine — 65 68

5. In Vitro Evaluation of the Anti-Inflammatory Effect and IntestinalPermeability Inhibitory Effect of Lactic Acid Bacteria

(1) Isolation of Dendritic Cells and Measurement of Inflammatory Marker

Immune cells were isolated from the bone marrow of C57BL/6 mice (male,20-23 g) by use of RPMI 1640 (containing 10% FBS, 1% antibiotics, 1%glutamax, 0.1% mercaptoethanol). The isolated cells were treated withRBC lysis buffer, washed, dispensed into each well of a 24-well plate,treated with GM-CSF and IL-4 at a ratio of 1:1000, and cultured. On 5days of the culturing, the medium was replaced with fresh medium, and on8 days, the cells were collected and used as dendritic cells. Next, thedendritic cells were seeded on a 24-well plate at a density of 0.5×10⁶cells/well and treated with lactic acid bacteria (test substance) andthe inflammation inducer LPS (lipopolysaccharide) for 2 hours or 24hours, and then the supernatant and the cells were collected. Using thecollected supernatant, the expression levels of IL-10 and IL-12 weremeasured by an immunoblotting method.

FIG. 4 is a graph showing the effect of lactic acid bacteria screened inthe present invention, on the lipopolysaccharide (LPS)-inducedinflammatory response of dendritic cells. The left graph in FIG. 4 showsthe effect of lactic acid bacteria on cells not treated with LPS(lipopolysaccharide), and the right graph shows the effect of lacticacid bacteria on cells treated with LPS (lipopolysaccharide). Inaddition, on the x-axis of FIG. 4, “Nor” indicates a group not treatedwith the test lactic acid bacteria and the inflammation inducer LPS(lipopolysaccharide); “LPS” indicates a group treated with theinflammation inducer LPS (lipopolysaccharide); “ch11” indicates a grouptreated with Lactobacillus sakei CH11; “ch15” indicates a group treatedwith Lactobacillus fermentum CH15; “ch23” indicates a group treated withLactobacillus brevis CH23; “ch32” indicates a group treated withLactobacillus johnsonii CH32; “ch38” indicates a group treated withBifidobacterium pseudocatenulatum CH38; “ch57” indicates a group treatedwith Bifidobacterium longum CH57; “ch57+ch11” indicates a group treatedwith a lactic acid bacteria mixture prepared by mixing Bifidobacteriumlongum CH57 and Lactobacillus sakei CH11 in the same amount; “ch57+ch23”indicates a group treated with a lactic acid bacteria mixture preparedby mixing Bifidobacterium longum CH57 and Lactobacillus brevis CH23 inthe same amount; and “ch57+ch32” indicates a group treated with a lacticacid bacteria mixture prepared by mixing Bifidobacterium longum CH57 andLactobacillus johnsonii CH32 in the same amount.

As shown in FIG. 4, Lactobacillus sakei CH11, Lactobacillus brevis CH23and Lactobacillus johnsonii CH32 induced IL-10 production of thedendritic cells obtained by differentiation after isolation from thebone marrow, effectively inhibited LPS (lipopolysaccharide)-inducedproduction of IL-12, and also the effects were increased when used incombination with Bifidobacterium longum CH57. In particular, a mixtureof Bifidobacterium longum CH57 and Lactobacillus brevis CH23 exhibitedthe best effect on the inhibition of inflammation. When dendritic cellsare controlled, Treg cells (regulatory T cells) can be efficientlycontrolled. For this reason, the lactic acid bacteria screened in thepresent invention can effectively alleviate chronic inflammatorydiseases such as colitis, autoimmune diseases such as rheumatoidarthritis and the like.

(2) Isolation of Macrophages and Measurement of Inflammatory Marker

6-week-old C57BL/6J male mice (20-23 g) were purchased from RaonBio Co.,Ltd. 2 ml of 4% sterile thioglycolate was administered into theabdominal cavity of each mouse, and after 96 hours, the mice wereanesthetized, and 8 ml of RPMI 1640 medium was administered into theabdominal cavity of each mouse. After 5 to 10 minutes, the RPMI medium(including macrophages) in the abdominal cavity of the mice was takenout, centrifuged at 1000 rpm for 10 minutes, and then washed twice withRPMI 1640 medium. The macrophages were seeded on a 24-well plate at adensity of 0.5×10⁶ cells/well and treated with the test substance lacticacid bacteria and the inflammation inducer LPS (lipopolysaccharide) for2 hours or 24 hours, and then the supernatant and the cells werecollected. The collected cells were homogenized in buffer (Gibco). Usingthe collected supernatant, the expression levels of cytokines such asTNF-α and IL-1β were measured by an ELISA kit. In addition, using thecollected cells, the expression levels of p65 (NF-kappa B), p-p65(phosphor-NF-kappa B) and β-actin were measured by an immunoblottingmethod. Specifically, 50 μg of the supernatant was taken andelectrophoresed on SDS 10% (w/v) polyacrylamide gel for 1 hour and 30minutes. The electrophoresed sample was transferred to a nitrocellulosemembrane under the conditions of 100 V and 400 mA for 1 hour and 10minutes. The sample-transferred nitrocellulose membrane was blocked with5% skim milk for 30 minutes, and then washed three times with PBS-Tweenfor 5 minutes each time, and incubated with a 1:100 dilution of primaryantibody (Santa Cruz Biotechnology, USA) overnight. Next, the membranewas washed three times for 10 minutes each time, and incubated with a1:1000 dilution of secondary antibody (Santa Cruz Biotechnology, USA)for 1 hour and 20 minutes. Next, the membrane was washed three times for15 minutes each time, and it was developed by fluorescence andvisualized.

FIG. 5 is a graph showing the effect of Bifidobacterium longum CH57 onthe LPS (lipopolysaccharide)-induced inflammatory response ofmacrophages. As shown in FIG. 5, Bifidobacterium longum CH57 effectivelyinhibited the LPS (lipopolysaccharide)-induced inflammatory response.

(3) Isolation of T Cells from Spleen and Measurement of Differentiationinto Th17 Cells or Treg Cells

Spleen was separated from C56BL/6J mice, crushed suitably, and suspendedin 10% FCS-containing RPMI 1640 medium, and CD4 T cells were isolatedtherefrom using a CD4 T cell isolation kit (MiltenyiBiotec, BergischGladbach, Germany). The isolated CD4 T cells were seeded in a 12-wellplate at a density of 5×10⁵ cells/well, and anti-CD3 (1 μg/ml,MiltenyiBiotec, Bergisch Gladbach, Germany) and anti-CD28 (1 μg/ml,MiltenyiBiotec, Bergisch Gladbach, Germany) were added thereto, oranti-CD3 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany),anti-CD28 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany),recombinant IL-6 (20 ng/ml, MiltenyiBiotec, Bergisch Gladbach, Germany)and recombinant transforming growth factor beta (1 ng/ml,MiltenyiBiotec, Bergisch Gladbach, Germany) were added. While the cellswere cultured, 1×10³ or 1×10⁵ CFU of the lactic acid bacteria were addedthereto, and the cells were cultured for 4 days. Next, the cells of theculture were stained with anti-FoxP3 or anti-IL-17A antibody, and thedistribution of Th17 and Treg cells was analyzed using a FACS(fluorescence-activated cell sorting) system (C6 Flow Cytometer® System,San Jose, Calif., USA).

FIG. 6 shows the results of analyzing the effect of Lactobacillus brevisCH23 on the differentiation of T cells (isolated from spleen) into Th17cells or Treg cells by a fluorescence-activated cell sorting system. Asshown in FIG. 6, Lactobacillus brevis CH23 inhibited the differentiationof T cells into Th17 cells (T helper 17 cells) and promoted thedifferentiation of T cells into Treg cells. These results suggest thatLactobacillus brevis CH23 can effectively alleviate inflammatorydiseases such as colitis and arthritis.

(4) Measurement of the Effect of Lactic Acid Bacteria on ZO-1 ProteinExpression of CaCO2 Cells

Caco2 cells obtained from the Korean Cell Line Bank were cultured inRPMI 1640 medium for 48 hours, and then the cultured Caco2 cells weredispensed into a 12-well plate at a density of 2×10⁶ cells/well. Next,each well was treated with 1 μg of LPS (lipopolysaccharide) alone or acombination of 1 μg of LPS (lipopolysaccharide) and 1×10³ CFU or 1×10⁵CFU of lactic acid bacteria, and then incubated for 24 hours. Next, thecultured cells were collected from each well, and the expression levelof tight junction protein ZO-1 was measured by an immunoblotting method.

FIG. 7 shows the results of analyzing the effect of Lactobacillus brevisCH23, Bifidobacterium longum CH57 or a mixture thereof on ZO-1 proteinexpression of CaCO2 cells. In FIG. 7, “CH23” indicates Lactobacillusbrevis CH23; “CH57” indicates Bifidobacterium longum CH57; “mix”indicates a lactic acid bacteria mixture prepared by mixingBifidobacterium longum CH57 and Lactobacillus johnsonii CH32 in the sameamount. As shown in FIG. 7, treatment with Lactobacillus brevis CH23 andBifidobacterium longum CH57 increased the expression of tight junctionprotein ZO-1, and treatment with a mixture of Bifidobacterium longumCH57 and Lactobacillus johnsonii CH32 synergistically increased theexpression of tight junction protein ZO-1. When the expression of tightjunction protein increases, in vivo penetration of toxic substances canbe blocked, thereby prevents the worsening of colitis, arthritis andliver injury.

6. In Vivo Evaluation of the Anti-Inflammatory and Colitis-AlleviatingEffects of Lactic Acid Bacteria

(1) Test Animals

5-Week-old C57BL/6 male mice (24-27 g) were purchased from OrientBio,and housed under controlled environmental conditions (humidity: 50±10%,temperature: 25±2° C., 12-hr light/12-hr dark cycle), and then used inthe experiment. As feed, standard experimental feed (Samyang, Korea) wasused, and the animals had access to drinking water ad libitum. In allthe experiments, one group consisted of 6 animals.

(2) Colitis Induction by TNBS and Sample Administration

One group of the test animals was used as a normal group, and the testanimals of the other groups were treated with2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce acute colitis.Specifically, the test animals were lightly anesthetized with ether, andthen a mixture solution of 2.5 g of TNBS (2,4,6-trinitrobenzene sulfonicacid) and 100 ml of 50% ethanol was administered into the colon throughthe anal in an amount of 0.1 ml each time by use of a 1-ml round-tipsyringe, lifted vertically and maintained for 30 seconds, therebyinducing inflammation. On the other hand, the normal group wasadministered orally with 0.1 ml of saline. On the next day, the lacticacid bacteria or the lactic acid bacteria mixture as a test sample wassuspended in saline and administered orally to each mouse in an amountof 2.0×10⁹ CFU, once a day for three days. On the next day following theend of sample administration, the animals were killed with carbondioxide, and a colon portion ranging from the cecum to the site justbefore the anus was dissected and used. Meanwhile, the test animals ofthe normal group were orally administered with saline alone instead ofthe lactic acid bacteria. In addition, the test animals of the negativecontrol group were orally administered with saline alone instead of thelactic acid bacteria after the induction of colitis by TNBS.Furthermore, the test animals of the positive control group were orallyadministered with 50 mg/kg of sulfasalazine, which is a drug fortreating colitis, instead of the lactic acid bacteria.

(3) Macroscopic Analysis of Colon

The length and appearance of the dissected colon were observed, and theappearance was analyzed by scoring according to the criteria (Hollenbachet al., 2005, Criteria for Degree of Colitis) shown in Table 9 below.After complete removal of colon contents, the colon tissue was washedwith saline. A portion of the washed colon tissue was fixed with 4%formaldehyde solution in order to use it as a pathological tissuesample, and the remainder was freeze-stored at −80° C. for molecularbiological analysis.

TABLE 9 Macroscopic score Criteria 0 Any ulcer and inflammation are notfound. 1 Edema without bleeding is found. 2 Ulcer with edema is found. 3Ulcer and inflammation are found at only one site. 4 Ulcer andinflammation are found at two or more sites. 5 Ulcer has an increasedsize of 2 cm or more.

(4) Measurement of Myeloperoxidase (MPO) Activity

100 mg of colon tissue was homogenized in 200 μl of 10 mM potassiumphosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammoniumbromide. The homogenized tissue was centrifuged at 10,000×g and 4° C.for 10 minutes, and the supernatant was collected. 50 μl of thesupernatant was added to 0.95 ml of a reaction solution (containing 1.6mM tetramethyl benzidine and 0.1 mM H₂O₂) and allowed to react at 37°C., and the absorbance at 650 nm was measured at various time pointsduring the reaction. To calculate myeloperoxidase (MPO) activity, 1μmol/ml of peroxide produced by the reaction was used as 1 unit.

(5) Measurement of Inflammatory Marker

Using a Western blotting method, inflammatory markers such as p-p65,p65, iNOS, COX-2 and β-actin were measured. Specifically, according tothe same method as the experiment for measurement of myeloperoxidase(MPO) activity, a supernatant was obtained. 50 μg of the supernatant wastaken and electrophoresed on SDS 10% (w/v) polyacrylamide gel for 1 hourand 30 minutes. The electrophoresed sample was transferred to anitrocellulose membrane under the conditions of 100 V and 400 mA for 1hour and 10 minutes. The sample-transferred nitrocellulose membrane wasblocked with 5% skim milk for 30 minutes, and then washed three timeswith PBS-Tween for 5 minutes each time, and incubated with a 1:100dilution of primary antibody (Santa Cruz Biotechnology, USA) overnight.Next, the membrane was washed three times for 10 minutes each time, andincubated with a 1:1000 dilution of secondary antibody (Santa CruzBiotechnology, USA) for 1 hour and 20 minutes. Next, the membrane waswashed three times for 15 minutes each time, and it was developed byfluorescence and visualized.

In addition, inflammation-related cytokines such as TNF-α, IL-1β and thelike were measured using an ELISA kit.

(6) Experimental Results

FIG. 8 shows the colon appearance or myeloperoxidase (MPO) activityindicating the effect of Bifidobacterium longum CH57 on model animalshaving acute colitis induced by TNBS; FIG. 9 depicts histological imagesof colon, which show the effect of Bifidobacterium longum CH57 on modelanimals having acute colitis induced by TNBS; and FIG. 10 showsinflammation-related cytokine levels indicating the effect ofBifidobacterium longum CH57 on model animals having acute colitisinduced by TNBS. In FIGS. 8 to 10, “NOR” indicates a normal group;“TNBS” indicates a negative control group; “CH57” indicates a groupadministered with Bifidobacterium longum CH57; and “SS50” indicates agroup administered with sulfasalazine. As shown in FIGS. 8 to 10,Bifidobacterium longum CH57 effectively alleviated colitis in view ofthe weight of the model animals having TNBS-induced acute colitis, thecolitis markers, the colon length, myeloperoxidase (MPO) activity, andthe like, and showed a better effect on the alleviation of colitis thansulfasalazine. In addition, Bifidobacterium longum CH57 inhibitedinflammatory cytokine production and increased the production of theanti-inflammatory cytokine IL-10 in the model animals havingTNBS-induced acute colitis.

FIG. 11 shows the colon appearance or myeloperoxidase (MPO) activityindicating the effect of Lactobacillus brevis CH23 on model animalshaving acute colitis induced by TNBS; FIG. 12 depicts histologicalimages of colon, which show the effect of Lactobacillus brevis CH23 onmodel animals having acute colitis induced by TNBS; FIG. 13 shows T-celldifferentiation patterns indicating the effect of Lactobacillus brevisCH23 on model animals having acute colitis induced by TNBS; and FIG. 14shows inflammation-related cytokine levels indicating the effect ofLactobacillus brevis CH23 on model animals having acute colitis inducedby TNBS. In FIGS. 11 to 14, “N” indicates a normal group; “TNBS”indicates a negative control group; “CH23” indicates a groupadministered with Lactobacillus brevis CH23; and “SS” indicates a groupadministered with sulfasalazine. As shown in FIGS. 11 to 14,Lactobacillus brevis CH23 effectively alleviated colitis in view of theweight of the model animals having TNBS-induced acute colitis, thecolitis markers, the colon length, myeloperoxidase (MPO) activity andthe like, and showed a better effect on the alleviation of colitis thansulfasalazine. In addition, Lactobacillus brevis CH23 inhibited thedifferentiation of T cells into Th17 cells and induced thedifferentiation of T cells into Treg cells in the model animals havingTNBS-induced acute colitis. Furthermore, Lactobacillus brevis CH23inhibited inflammatory cytokine production and increased the productionof the anti-inflammatory cytokine IL-10 in the model animals havingTNBS-induced acute colitis.

FIG. 15 shows the colon appearance or myeloperoxidase (MPO) activityindicating the effect of a mixture of Bifidobacterium longum CH57 andLactobacillus brevis CH23 on model animals having acute colitis inducedby TNBS; FIG. 16 depicts histological images showing the effect of amixture of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 onmodel animals having acute colitis induced by TNBS; and FIG. 17 showsinflammation-related cytokine levels indicating the effect of a mixtureof Bifidobacterium longum CH57 and Lactobacillus brevis CH23 on modelanimals having acute colitis induced by TNBS. In FIGS. 15 to 17, “NOR”indicates a normal group; “TNBS” indicates a negative control group;“BL” indicates a group administered with a lactic acid bacteria mixtureprepared by mixing Bifidobacterium longum CH57 and Lactobacillus brevisCH23 in the same amount; and “SS50” indicates a group administered withsulfasalazine. As shown in FIGS. 15 to 17, a lactic acid bacteriamixture of Bifidobacterium longum CH57 and Lactobacillus brevis CH23significantly improved effects against the reduced weight of the modelanimals having TNBS-induced acute colitis, increased colitis markerlevels, shortened colon lengths and increased myeloperoxidase (MPO)activity, and the effect thereof on the alleviation of colitis wassignificantly better than that of sulfasalazine. In addition, the lacticacid bacteria mixture of Bifidobacterium longum CH57 and Lactobacillusbrevis CH23 significantly inhibited inflammatory cytokine production anddramatically increased the production of anti-inflammatory cytokineIL-10 in the model animals having TNBS-induced acuter colitis.

7. In Vivo Evaluation of the Obesity-Reducing and Anti-InflammatoryEffects of Lactic Acid Bacteria

(1) Experimental Method

A total of 24 C57BL6/J mice were purchased from RaonBio Co., Ltd., andacclimated with chow diet (Purina) under the conditions of temperatureof 20±2° C., humidity of 50±10% and 12-hr light/12-hr dark cycle for 1week. Next, the test animals were divided into three groups (LFD, HFD,and HFD+BL), each consisting of 8 animals, the LFD group was fed with anormal diet (LFD, 10% of calories from fat; Research, NJ, USA) for 4weeks, and the HFD group and the HFD+BL group were fed with a high-fatdiet (HFD, 60% of calories from fat; Research, NJ, USA) for 4 weeks.Next, the LFD group was orally administered with PBS while fed with thenormal diet for 4 weeks. Furthermore, the HFD group was orallyadministered with PBS while fed with the high-fat diet for 4 weeks. Inaddition, the HFD+BL group was orally administered with a PBS suspensionof 2×10⁹ CFU of a lactic acid bacteria mixture while fed with thehigh-fat diet for 4 weeks. The lactic acid bacteria mixture was preparedby mixing Bifidobacterium longum CH57 and Lactobacillus brevis CH23 inthe same amount.

(2) Analysis of the Anti-Obesity Effect and Anti-Inflammatory Effect ofLactic Acid Bacteria Mixture

The anti-obesity effect of the lactic acid bacteria mixture was analyzedthrough weight change. In addition, the anti-inflammatory effect of thelactic acid bacteria mixture was analyzed using the same method as thatused in the experiment on the model animals having TNBS-induced acutecolitis.

(3) Experimental Results

FIG. 18 shows weight changes indicating the effect of a mixture ofBifidobacterium longum CH57 and Lactobacillus brevis CH23 onobesity-induced model animals; FIG. 19 shows the appearance of colon,myeloperoxidase (MPO) activity, histological images of colon and thelike, which indicate the effect of a mixture of Bifidobacterium longumCH57 and Lactobacillus brevis CH23 on obesity-induced model animals;FIG. 20 shows inflammation-related cytokine levels indicating the effectof a mixture of Bifidobacterium longum CH57 and Lactobacillus brevisCH23 on obesity-induced model animals; and FIG. 21 shows inflammatoryresponse markers indicating the effect of a mixture of Bifidobacteriumlongum CH57 and Lactobacillus brevis CH23 on obesity-induced modelanimals. As shown in FIGS. 18 to 21, the lactic acid bacteria mixture ofBifidobacterium longum CH57 and Lactobacillus brevis CH23 greatlyreduced the increased weight, increased colitis marker levels andincreased myeloperoxidase (MPO) activity of the model animals havingobesity induced by the high-fat diet, and inhibited the development ofcolitis. In addition, the lactic acid bacteria mixture ofBifidobacterium longum CH57 and Lactobacillus brevis CH23 greatlyinhibited inflammatory cytokine production and increased the productionof anti-inflammatory cytokine IL-10 in the model animals having obesityinduced by the high-fat diet.

II. Second Experiment for Screening of Lactic Acid Bacteria andEvaluation of the Effects Thereof

1. Isolation and Identification of Lactic Acid Bacteria

(1) Isolation of Lactic Acid Bacteria from Kimchi

Each of Chinese cabbage kimchi, radish kimchi and green onion kimchi wascrushed, and the crushed liquid was suspended in MRS liquid medium (MRSBroth; Difco, USA). Next, the supernatant was collected, transferred toMRS agar medium (Difco, USA), and cultured anaerobically at 37° C. forabout 48 hours, and then Bifidobacterium longum strains that formedcolonies were separated according to shape.

(2) Isolation of Lactic Acid Bacteria from Human Feces

Human feces were suspended in GAM liquid medium (GAM broth; NissuiPharmaceutical, Japan). Next, the supernatant was collected, transferredto BL agar medium (Nissui Pharmaceutical, Japan) and culturedanaerobically at 37° C. for about 48 hours, and then Bifidobacterium sp.strains that formed colonies were isolated.

(3) Identification of Screened Lactic Acid Bacteria

The gram-staining characteristics, physiological characteristics and 16SrDNA sequences of the strains isolated from kimchi or human feces wereanalyzed to identify the species of the strains, and names were given tothe strains. Table 10 below the control numbers and strain names of thelactic acid bacteria isolated from Chinese cabbage kimchi, radish kimchiand green onion kimchi, and Table 11 below shows the control numbers andstrain names of the lactic acid bacteria isolated from human feces.

TABLE 10 Control No. Strain name 1 Lactobacillus plantarum LC1 2Lactobacillus plantarum LC2 3 Lactobacillus plantarum LC3 4Lactobacillus plantarum LC4 5 Lactobacillus plantarum LC5 6Lactobacillus plantarum LC6 7 Lactobacillus plantarum LC7 8Lactobacillus plantarum LC8 9 Lactobacillus plantarum LC9 10Lactobacillus plantarum LC10 11 Lactobacillus plantarum LC11 12Lactobacillus plantarum LC12 13 Lactobacillus plantarum LC13 14Lactobacillus plantarum LC14 15 Lactobacillus plantarum LC15 16Lactobacillus plantarum LC16 17 Lactobacillus plantarum LC17 18Lactobacillus plantarum LC18 19 Lactobacillus plantarum LC19 20Lactobacillus plantarum LC20 21 Lactobacillus plantarum LC21 22Lactobacillus plantarum LC22 23 Lactobacillus plantarum LC23 24Lactobacillus plantarum LC24 25 Lactobacillus plantarum LC25 26Lactobacillus plantarum LC26 27 Lactobacillus plantarum LC27 28Lactobacillus plantarum LC28 29 Lactobacillus plantarum LC29 30Lactobacillus plantarum LC30 31 Lactobacillus plantarum LC31 32Lactobacillus plantarum LC32 33 Lactobacillus plantarum LC33 34Lactobacillus plantarum LC34 35 Lactobacillus plantarum LC35 36Lactobacillus plantarum LC36 37 Lactobacillus plantarum LC37 38Lactobacillus plantarum LC38 39 Lactobacillus plantarum LC39 40Lactobacillus plantarum LC40 41 Lactobacillus plantarum LC41 42Lactobacillus plantarum LC42 43 Lactobacillus plantarum LC43 44Lactobacillus plantarum LC44 45 Lactobacillus plantarum LC45 46Lactobacillus plantarum LC46 47 Lactobacillus plantarum LC47 48Lactobacillus plantarum LC48 49 Lactobacillus plantarum LC49 50Lactobacillus plantarum LC50

TABLE 11 Control No. Strain name 51 Bifidobacterium longum LC51 52Bifidobacterium longum LC52 53 Bifidobacterium longum LC53 54Bifidobacterium longum LC54 55 Bifidobacterium longum LC55 56Bifidobacterium longum LC56 57 Bifidobacterium longum LC57 58Bifidobacterium longum LC58 59 Bifidobacterium longum LC59 60Bifidobacterium longum LC60 61 Bifidobacterium longum LC61 62Bifidobacterium longum LC62 63 Bifidobacterium longum LC63 64Bifidobacterium longum LC64 65 Bifidobacterium longum LC65 66Bifidobacterium longum LC66 67 Bifidobacterium longum LC67 68Bifidobacterium longum LC68 69 Bifidobacterium longum LC69 70Bifidobacterium longum LC70 71 Bifidobacterium longum LC71 72Bifidobacterium longum LC72 73 Bifidobacterium longum LC73 74Bifidobacterium longum LC74 75 Bifidobacterium longum LC75 76Bifidobacterium longum LC76 77 Bifidobacterium longum LC77 78Bifidobacterium longum LC78 79 Bifidobacterium longum LC79 80Bifidobacterium longum LC80 81 Bifidobacterium longum LC81 82Bifidobacterium longum LC82 83 Bifidobacterium longum LC83 84Bifidobacterium longum LC84 85 Bifidobacterium longum LC85 86Bifidobacterium longum LC86 87 Bifidobacterium longum LC87 88Bifidobacterium longum LC88 89 Bifidobacterium longum LC89 90Bifidobacterium longum LC90 91 Bifidobacterium longum LC91 92Bifidobacterium longum LC92 93 Bifidobacterium longum LC93 94Bifidobacterium longum LC94 95 Bifidobacterium longum LC95 96Bifidobacterium longum LC96 97 Bifidobacterium longum LC97 98Bifidobacterium longum LC98 99 Bifidobacterium longum LC99 100Bifidobacterium longum LC100

It was shown that Lactobacillus plantarum LC5 shown in Table 10 abovewas a gram-positive anaerobic bacillus and the 16S rDNA thereof had anucleotide sequence of SEQ ID NO: 4. The 16S rDNA nucleotide sequence ofLactobacillus plantarum LC5 was identified by BLAST in Genebank(http://www.ncbi.nlm.nih.gov/), and as a result, a Lactobacillusplantarum strain having the same 16S rDNA nucleotide sequence as that ofLactobacillus plantarum LC5 was not found, and Lactobacillus plantarumLC5 showed a homology of 99% with the 16S rDNA sequence of Lactobacillusplantarum strain KF9. Furthermore, it was shown that Lactobacillusplantarum LC27 shown in Table 10 above was a gram-positive anaerobicbacillus and the 16S rDNA thereof had a nucleotide sequence of SEQ IDNO: 5. The 16S rDNA nucleotide sequence of Lactobacillus plantarum LC27was identified by BLAST in Genebank (http://www.ncbi.nlm.nih.gov/), andas a result, a Lactobacillus plantarum strain having the same 16S rDNAnucleotide sequence as that of Lactobacillus plantarum LC27 was notfound, and Lactobacillus plantarum LC27 showed a homology of 99% withthe 16S rDNA sequence of Lactobacillus plantarum strain JL18. Inaddition, it was shown that Lactobacillus plantarum LC28 shown in Table10 above was a gram-positive anaerobic bacillus and the 16S rDNA thereofhad a nucleotide sequence of SEQ ID NO: 6. The 16S rDNA nucleotidesequence of Lactobacillus plantarum LC28 was identified by BLAST inGenebank (http://www.ncbi.nlm.nih.gov/), and as a result, aLactobacillus plantarum strain having the same 16S rDNA nucleotidesequence as that of Lactobacillus plantarum LC28 was not found, andLactobacillus plantarum LC28 showed a homology of 99% with the 16S rDNAsequence of Lactobacillus plantarum strain USIM01.

It was shown that Bifidobacterium longum LC67 shown in Table 11 abovewas a gram-positive anaerobic bacillus and the 16S rDNA thereof had anucleotide sequence of SEQ ID NO: 7. The 16S rDNA nucleotide sequence ofBifidobacterium longum LC67 was identified by BLAST in Genebank(http://www.ncbi.nlm.nih.gov/), and as a result, a Bifidobacteriumlongum strain having the same 16S rDNA nucleotide sequence as that ofBifidobacterium longum LC67 was not found, and Bifidobacterium longumLC67 showed a homology of 99% with the 16S rDNA sequence ofBifidobacterium longum strain CBT-6. Furthermore, it was shown thatBifidobacterium longum LC68 shown in Table 11 above was a gram-positiveanaerobic bacillus and the 16S rDNA thereof had a nucleotide sequence ofSEQ ID NO: 8. The 16S rDNA nucleotide sequence of Bifidobacterium longumLC68 was identified by BLAST in Genebank (http://www.ncbi.nlm.nih.gov/),and as a result, a Bifidobacterium longum strain having the same 16SrDNA nucleotide sequence as that of Bifidobacterium longum LC68 was notfound, and Bifidobacterium longum LC68 showed a homology of 99% with the16S rDNA sequence of Bifidobacterium longum strain IMAUFB067.

In addition, among the physiological characteristics of Lactobacillusplantarum LC5, Lactobacillus plantarum LC27, Bifidobacterium longum LC67and Bifidobacterium longum LC68, the carbon source utilization wasanalyzed using a sugar fermentation by an API kit (model: API 50 CHL;manufactured by BioMerieux's, USA). Table 12 below shows the results ofanalyzing the carbon source utilization of Lactobacillus plantarum LC5and Lactobacillus plantarum LC27, and Table 13 below shows the resultsof analyzing the carbon source utilization of Bifidobacterium longumLC67 and Bifidobacterium longum LC68. In Tables 12 and 13 below, “+”indicates the case in which carbon source utilization is positive; “−”indicates the case in which carbon source utilization is negative; and“±” indicates the case in which carbon source utilization is ambiguous.As shown in 12 and 13 below, Lactobacillus plantarum LC5, Lactobacillusplantarum LC27, Bifidobacterium longum LC67 and Bifidobacterium longumLC68 showed carbon source utilization different from that of knownstrains of the same species with respect to some carbon sources.

TABLE 12 Strain name Strain name L. plantarum L. plantarum L. plantarumL. plantarum Carbon source LC5 LC27 Carbon source LC5 LC27 glycerol − −salicin + + erythritol − − cellobiose + + D-arabinose − − maltose + +L-arabinose − + lactose − + D-ribose + + melibiose + + D-xylose − −sucrose + + L-xylose − − trehalose + + D-adonitol − − inulin − −methyl-β-D-xylopyranoside − − melezitose + + D-galactose + ± raffinose ±− D-glucose + + starch − − D-fructose + + glycogen − − D-mannose + +xylitol − − L-sorbose − − gentiobiose + + L-rhamnose − ± D-turanose − +dulcitol − − D-lyxose − − inositol − − D-tagatose − − mannitol + +D-fucose − − sorbitol + + L-fucose − − α-methyl-D-mannoside − ±D-arabitol − − α-methly-D-glucoside − − L-arabitol − −N-acetyl-glucosamine + + gluconate − − amygdalin + + 2-keto-gluconate −− arbutin + + 5-keto-gluconate − − esculin + +

TABLE 13 Strain name Strain name B. longum B. longum B. longum B. longumCarbon source LC67 LC68 Carbon source LC67 LC68 glycerol − − salicin − −erythritol − − cellobiose − − D-arabinose − − maltose + +L-arabinose + + lactose + + D-ribose − − melibiose + + D-xylose + ±sucrose + ± L-xylose − − trehalose − ± D-adonitol − − inulin − −methyl-β-D-xylopyranoside − − melezitose − + D-galactose ± +raffinose + + D-glucose + + starch − − D-fructose + + glycogen − −D-mannose − − xylitol − − L-sorbose − − gentiobiose + ± L-rhamnose − −D-turanose ± ± dulcitol − − D-lyxose − − inositol − − D-tagatose − −mannitol ± + D-fucose − − sorbitol ± + L-fucose − − α-methyl-D-mannoside− − D-arabitol − − α-methly-D-glucoside ± ± L-arabitol − −N-acetyl-glucosamine − − gluconate − − amygdalin − ± 2-keto-gluconate −− arbutin − − 5-keto-gluconate − − esculin + +

(4) Information on Deposition of Lactic Acid Bacteria

The present inventors deposited Lactobacillus plantarum LC5 with theKorean Culture Center of Microorganisms (address: Yurim Building, 45,Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an internationaldepositary authority, on Jan. 11, 2016 under accession number KCCM11800P. Furthermore, the present inventors deposited Lactobacillusplantarum LC27 with the Korean Culture Center of Microorganisms(address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul,Korea), an international depositary authority, on Jan. 11, 2016 underaccession number KCCM 11801P. Furthermore, the present inventorsdeposited Bifidobacterium longum LC67 with the Korean Culture Center ofMicroorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil,Seodaemun-gu, Seoul, Korea), an international depositary authority, onJan. 11, 2016 under accession number KCCM 11802P.

2. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation ofIntestinal Damage or Intestinal Permeability

In order to evaluate the effect of the lactic acid bacteria isolatedfrom kimchi or human feces, on the alleviation of intestinal damage orinternal permeability, the antioxidant activity, lipopolysaccharide(LPS) production inhibitory activity, β-glucuronidase (harmfulintestinal enzyme) inhibitory activity and tight junction proteinexpression-inducing activity of the lactic acid bacteria were measured.

(1) Experimental Methods

*Antioxidant Activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in ethanol to aconcentration of 0.2 mM to prepare a DPPH solution. A lactic acidbacteria suspension (1×10⁸ CFU/ml) or a vitamin C solution (1 g/ml) wasadded to 0.1 ml of the DPPH solution and cultured at 37° C. for 20minutes. The culture was centrifuged at 3000 rpm for 5 minutes, and thesupernatant was collected. Next the absorbance of the supernatant at 517nm was measured, and the antioxidant activity of the lactic acidbacteria was calculated.

*Lipopolysaccharide (LPS) Production Inhibitory Activity

0.1 g of human fresh feces was suspended in 0.9 ml of sterilephysiological saline and diluted 100-fold with general anaerobic mediumto prepare a fecal suspension. 0.1 ml of the fecal suspension and 0.1 mlof lactic acid bacteria (1×10⁴ or 1×10⁵ CFU) were added to 9.8 ml ofsterile anaerobic medium (Nissui Pharmaceuticals, Japan) and culturedanaerobically for 24 hours. Next, the culture was sonicated for about 1hour to disrupt the outer cell membrane of the bacteria, and centrifugedat 5000×g and the supernatant was collected. Next, the content of LPS(lipopolysaccharide) (which is a typical endotoxin) in the supernatantwas measured by a LAL (Limulus Amoebocyte Lysate) assay kit(manufactured by Cape Cod Inc., USA). In addition, in order to evaluatethe E. coli proliferation inhibitory activity of the lactic acidbacteria, the culture obtained through the same experiment as describedabove was diluted 1000-fold and 100000-fold and cultured in DHL medium,and then the number of E. coli cells was counted.

*β-Glucuronidase Inhibitory Activity

0.1 ml of 0.1 mM p-nitrophenyl-β-D-glucuronide solution, 0.2 ml of 50 mMphosphate buffered saline and 0.1 ml of a lactic acid bacteriasuspension (prepared by suspending of a lactic acid bacteria culture in5 ml of physiological saline) were placed in a reactor and subjected toβ-glucuronidase enzymatic reaction, and 0.5 ml of 0.1 mM NaOH solutionwas added to stop the reaction. Next, the reaction solution wascentrifuged at 3000 rpm for 5 minutes, and the supernatant wascollected. Then, the absorbance of the supernatant at 405 nm wasmeasured.

*Tight Junction Protein Expression-Inducing Activity

Caco2 cells obtained from the Korean Cell Line Bank were cultured inRPMI 1640 medium for 48 hours, and then the cultured Caco2 cells weredispensed to each well of a 12-well plate at a density of 2×10⁶cells/well. Next, each well was treated with 1 μg of LPS(lipopolysaccharide) or a combination of 1 μg of LPS(lipopolysaccharide) and 1×10³ CFU of lactic acid bacteria and incubatedfor 24 hours. Next, the cultured cells were collected from each well,and the expression level of tight junction protein ZO-1 in the cells wasmeasured by an immunoblotting method.

(2) Experimental Results

The antioxidant activity, lipopolysaccharide (LPS) production inhibitoryactivity, β-glucuronidase inhibitory activity and tight junction proteinexpression-inducing activity of the lactic acid bacteria isolated fromkimchi or human feces were measured, and the results of the measurementare shown in Tables 14 to 16 below. As shown in Tables 14 to 16 below,Lactobacillus plantarum LC5, Lactobacillus plantarum LC15, Lactobacillusplantarum LC17, Lactobacillus plantarum LC25, Lactobacillus plantarumLC27, Lactobacillus plantarum LC28, Bifidobacterium longum LC55,Bifidobacterium longum LC65, Bifidobacterium longum LC67 andBifidobacterium longum LC68 had excellent antioxidant activity, stronglyinhibited lipopolysaccharide (LPS) production and β-glucuronidaseactivity, and strongly induced the expression of tight junction protein.In particular, Bifidobacterium longum LC67 showed the best tightjunction protein expression-inducing activity. These lactic acidbacteria have an excellent antioxidant effect, have an excellent effectof inhibiting the enzymatic activity of intestinal flora's harmfulbacteria associated with inflammation and carcinogenesis, inhibit theproduction of endotoxin LPS (lipopolysaccharide) produced by intestinalflora's harmful bacteria, and induce the expression of tight junctionprotein. Thus, these lactic acid bacteria can improve intestinalpermeability syndrome.

TABLE 14 Tight junction protein Beta- LPS production expression ControlAntioxidant glucuronidase inhibitory inducing No. Strain name activityinhibitory activity activity activity 1 Lactobacillus plantarum LC1++ + + − 2 Lactobacillus plantarum LC2 ++ ++ + − 3 Lactobacillusplantarum LC3 +++ ++ + − 4 Lactobacillus plantarum LC4 +++ ++ + + 5Lactobacillus plantarum LC5 +++ +++ ++ ++ 6 Lactobacillus plantarum LC6++ +++ + − 7 Lactobacillus plantarum LC7 +++ ++ + − 8 Lactobacillusplantarum LC8 ++ +++ + − 9 Lactobacillus plantarum LC9 ++ ++ + + 10Lactobacillus plantarum LC10 ++ +++ + + 11 Lactobacillus plantarum LC11++ ++ + − 12 Lactobacillus plantarum LC12 ++ +++ + + 13 Lactobacillusplantarum LC13 ++ ++ + + 14 Lactobacillus plantarum LC14 ++ ++ + − 15Lactobacillus plantarum LC15 +++ +++ ++ ++ 16 Lactobacillus plantarumLC16 + +++ + − 17 Lactobacillus plantarum LC17 +++ +++ ++ ++ 18Lactobacillus plantarum LC18 ++ ++ + + 19 Lactobacillus plantarum LC19++ +++ + + 20 Lactobacillus plantarum LC20 ++ ++ + − 21 Lactobacillusplantarum LC21 ++ ++ + − 22 Lactobacillus plantarum LC22 ++ +++ − − 23Lactobacillus plantarum LC23 +++ ++ − − 24 Lactobacillus plantarum LC24+++ + − − 25 Lactobacillus plantarum LC25 +++ +++ ++ ++ 26 Lactobacillusplantarum LC26 ++ + + + 27 Lactobacillus plantarum LC27 +++ +++ ++ ++ 28Lactobacillus plantarum LC28 +++ +++ ++ ++ 29 Lactobacillus plantarumLC29 ++ + − − 30 Lactobacillus plantarum LC30 ++ + + − 31 Lactobacillusplantarum LC31 +++ ++ + − 32 Lactobacillus plantarum LC32 +++ ++ + − 33Lactobacillus plantarum LC33 +++ ++ + − 34 Lactobacillus plantarum LC34++ ++ + + 35 Lactobacillus plantarum LC35 ++ ++ + +

TABLE 15 Tight junction protein Beta- LPS production expression ControlAntioxidant glucuronidase inhibitory inducing No. Strain name activityinhibitory activity activity activity 36 Lactobacillus plantarum LC36 ++++ ++ − 37 Lactobacillus plantarum LC37 +++ ++ + + 38 Lactobacillusplantarum LC38 ++ ++ + − 39 Lactobacillus plantarum LC39 ++ + + − 40Lactobacillus plantarum LC40 +++ + − − 41 Lactobacillus plantarum LC41++ ++ − + 42 Lactobacillus plantarum LC42 +++ + − + 43 Lactobacillusplantarum LC43 ++ + − + 44 Lactobacillus plantarum LC44 ++ + − + 45Lactobacillus plantarum LC45 ++ ++ − + 46 Lactobacillus plantarum LC46++ + − + 47 Lactobacillus plantarum LC47 +++ + − + 48 Lactobacillusplantarum LC48 ++ ++ − + 49 Lactobacillus plantarum LC49 ++ +++ + + 50Lactobacillus plantarum LC50 +++ ++ + − 51 Bifidobacterium longum LC51++ ++ + − 52 Bifidobacterium longum LC52 +++ +++ + − 53 Bifidobacteriumlongum LC53 ++ +++ − + 54 Bifidobacterium longum LC54 +++ ++ + + 55Bifidobacterium longum LC55 +++ +++ ++ ++ 56 Bifidobacterium longum LC56+++ ++ + + 57 Bifidobacterium longum LC57 ++ + − + 58 Bifidobacteriumlongum LC58 +++ + − + 59 Bifidobacterium longum LC59 ++ + − − 60Bifidobacterium longum LC60 +++ + − − 61 Bifidobacterium longum LC61++ + + − 62 Bifidobacterium longum LC62 +++ + + − 63 Bifidobacteriumlongum LC63 ++ ++ ++ − 64 Bifidobacterium longum LC64 +++ + − − 65Bifidobacterium longum LC65 +++ +++ ++ ++ 66 Bifidobacterium longum LC66++ + + + 67 Bifidobacterium longum LC67 +++ +++ ++ +++ 68Bifidobacterium longum LC68 +++ +++ ++ ++ 69 Bifidobacterium longum LC69+++ + − − 70 Bifidobacterium longum LC70 ++ + − +

TABLE 16 Tight junction protein Beta- LPS production expression ControlAntioxidant glucuronidase inhibitory inducing No. Strain name activityinhibitory activity activity activity 71 Bifidobacterium longum LC71++ + − + 72 Bifidobacterium longum LC72 +++ ++ − + 73 Bifidobacteriumlongum LC73 ++ ++ + − 74 Bifidobacterium longum LC74 ++ +++ + − 75Bifidobacterium longum LC75 +++ + − + 76 Bifidobacterium longum LC76++ + − + 77 Bifidobacterium longum LC77 ++ ++ + + 78 Bifidobacteriumlongum LC78 ++ + + + 79 Bifidobacterium longum LC79 +++ + + + 80Bifidobacterium longum LC80 ++ + + + 81 Bifidobacterium longum LC81++ + + + 82 Bifidobacterium longum LC82 ++ ++ − + 83 Bifidobacteriumlongum LC83 +++ + − + 84 Bifidobacterium longum LC84 ++ ++ − − 85Bifidobacterium longum LC85 +++ ++ − + 86 Bifidobacterium longum LC86++ + + − 87 Bifidobacterium longum LC87 ++ ++ + − 88 Bifidobacteriumlongum LC88 ++ +++ + + 89 Bifidobacterium longum LC89 ++ ++ + + 90Bifidobacterium longum LC90 ++ ++ + + 91 Bifidobacterium longum LC91 ++++++ + + 92 Bifidobacterium longum LC92 +++ ++ + + 93 Bifidobacteriumlongum LC93 ++ ++ + + 94 Bifidobacterium longum LC94 ++ ++ − + 95Bifidobacterium longum LC95 ++ +++ − − 96 Bifidobacterium longum LC96++ + − − 97 Bifidobacterium longum LC97 ++ + − − 98 Bifidobacteriumlongum LC98 ++ ++ − − 99 Bifidobacterium longum LC99 ++ ++ − − 100Bifidobacterium longum LC100 ++ ++ − + * The final concentration oflactic acid bacteria in measurement of antioxidant activity: 1 × 10⁴CFU/ml; the concentration of lactic acid bacteria added for measurementof beta-glucuronidase inhibitory activity and lipopolysaccharide (LPS)production inhibitory activity: 1 × 10⁴ CFU/ml; the concentration oflactic acid bacteria in measurement of tight junction proteinexpression-inducing activity: 1 × 10⁴ CFU/ml. * Criteria for measurementof various activities of lactic acid bacteria: very strongly(+++; >90%); strongly (++; >60-90%); weakly (+; >20-60%); not or lessthan 20% (−; <20%).

3. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation ofLiver Injury

Based on evaluation of the effect of the lactic acid bacteria on thealleviation of intestinal damage or intestinal permeability syndrome,the following ten strains were selected: Lactobacillus plantarum LC5,Lactobacillus plantarum LC15, Lactobacillus plantarum LC17,Lactobacillus plantarum LC25 Lactobacillus plantarum LC27, Lactobacillusplantarum LC28, Bifidobacterium longum LC55, Bifidobacterium longumLC65, Bifidobacterium longum LC67 and Bifidobacterium longum LC68. Theeffect of each of these selected lactic acid bacteria strains or amixture of these strains on the alleviation of liver injury wasevaluated using model animals having liver injury induced bytert-butylperoxide.

1) Experimental Method

Mice (C57BL/6, male) were divided into several groups, each consistingof 6 animals. Tert-butylperoxide was administered intraperitoneally tothe test animals of groups other than a normal group at a dose of 2.5mmol/kg to induce liver injury. From 2 hours after administration oftert-butylperoxide, 2×10⁹ CFU of lactic acid bacteria were administeredorally to the test animals of groups other than the normal group and thenegative control group, once a day for days. In addition, silymarin inplace of lactic acid bacteria was administered orally to the testanimals of the positive control group at a dose of 100 mg/kg, once a dayfor 3 days. At 6 hours after the last administration of the drug, bloodwas taken from the heart. The taken blood was allowed to stand at roomtemperature for 60 minutes, and centrifuged at 3,000 rpm for 15 minutesto separate serum. The GPT (glutamic pyruvate transaminase) and GOT(glutamic oxalacetic transaminase) levels in the separated serum weremeasured using a blood assay kit (ALT & AST measurement kit; Asan Pharm.Co., Korea). In addition, 1 g of the liver tissue dissected from eachtest animal was added to saline and homogenized using a homogenizer, andthe supernatant was analyzed by an ELISA kit to measure the level ofTNF-α.

(2) Experimental Results

Table 17 below shows the changes in GOT, GPT and TNF-α values whenlactic acid bacteria were administered to model animals having liverinjury induced by tert-butylperoxide. As shown in Table 17 below,Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillusplantarum LC28, Bifidobacterium longum LC67 and Bifidobacterium longumLC68 showed excellent effects on the alleviation of liver injurycompared to silymarin, and mixtures of these lactic acid bacteria showedbetter effects on the alleviation of liver injury.

TABLE 17 GOT GPT TNF-α Test groups (IU/L) (IU/L) (pg/g) Normal group42.4 6.2 140.4 Negative control group 103.1 28.0 298.0 Groupadministered with LC5 36.9 5.4 115.7 Group administered with LC15 60.36.2 154.3 Group administered with LC17 65.8 6.8 136.7 Group administeredwith LC25 64.6 11.3 132.4 Group administered with LC27 35.3 3.3 157.1Group administered with LC28 42.0 1.0 185.7 Group administered with LC5555.6 17.6 251.4 Group administered with LC65 61.4 17.3 127.6 Groupadministered with LC67 50.8 3.8 150.5 Group administered with LC68 40.85.7 82.4 Group administered with LC5 + LC67 32.7 3.1 115.9 Groupadministered with LC5 + LC68 36.8 5.6 105.4 Group administered withLC27 + LC67 30.5 2.3 121.2 Group administered with LC27 + LC68 35.4 3.2112.8 Group administered with LC28 + LC67 32.5 2.8 128.2 Groupadministered with silymarin 52.9 5.9 93.8

In Table 17 above, “LC5” indicates Lactobacillus plantarum LC5; “LC15”indicates Lactobacillus plantarum LC15; “LC17” indicates Lactobacillusplantarum LC17; “LC25” indicates Lactobacillus plantarum LC25; “LC27”indicates Lactobacillus plantarum LC27; “LC28” indicates Lactobacillusplantarum LC28; “LC55” indicates Bifidobacterium longum LC55; “LC65”indicates Bifidobacterium longum LC65; “LC67” indicates Bifidobacteriumlongum LC67; “LC68” indicates Bifidobacterium longum LC68; “LC5+LC67”indicates a lactic acid bacteria mixture prepared by mixingLactobacillus plantarum LC5 and Bifidobacterium longum LC67 in the sameamount; “LC5+LC68” indicates a lactic acid bacteria mixture prepared bymixing Lactobacillus plantarum LC5 and Bifidobacterium longum LC68 inthe same amount; “LC27+LC67” indicates a lactic acid bacteria mixtureprepared by mixing Lactobacillus plantarum LC27 and Bifidobacteriumlongum LC67 in the same amount; “LC27+LC68” indicates a lactic acidbacteria mixture prepared by mixing Lactobacillus plantarum LC27 andBifidobacterium longum LC68 in the same amount; and “LC28+LC67”indicates a lactic acid bacteria mixture prepared by mixingLactobacillus plantarum LC28 and Bifidobacterium longum LC67 in the sameamount. In the following Tables showing the experimental results, thesame symbols are used for single lactic acid bacteria or lactic acidbacteria mixtures.

4. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation ofAllergy

(1) Measurement of the Inhibition of Degranulation by Lactic AcidBacteria

The RBL-2H3 cell line (rat mast cell line, the Korean Cell Line Bank,Cat. No. 22256) was cultured with DMEM (Dulbeccos' modified Eagle'smedium, Sigma, 22256) containing 10% FBS (fetal bovine serum) andL-glutamine in a humidified 5% CO₂ incubator at 37° C. The cellscontained in the culture medium were floated using trypsin-EDTAsolution, and the floated cells were isolated, collected and used in theexperiment. The collected RBL-2H3 cells were dispensed into a 24-wellplate at a density of 5×10⁵ cells/well and sensitized by incubation with0.5 μg/ml of mouse monoclonal IgE for 12 hours. The sensitized cellswere washed with 0.5 ml of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4mM MgCl₂, 25 mM PIPES, 40 mM NaOH, pH 7.2), and then incubated with 0.16ml of siraganian buffer (supplemented with 5.6 mM glucose, 1 mM CaCl₂,0.1% BSA) at 37° C. for 10 minutes. Next, lactic acid bacteria as a testdrug were added to the cell culture to a concentration of 1×10⁴ CFU/ml,or 0.04 ml of DSCG (disodium cromoglycate) as a control drug was addedto the cell culture, and after 20 minutes, the cells were activated with0.02 ml of antigen (1 μg/ml DNP-BSA) at 37° C. for 10 minutes. Next, thecell culture was centrifuged at 2000 rpm for 10 minutes, and thesupernatant was collected. 0.025 ml of the collected supernatant wastransferred to a 96-well plate, and then 0.025 ml of 1 mM p-NAG (asolution of p-nitrophenyl-N-acetyl-β-D-glucosamide in 0.1M citratebuffer, pH 4.5) was added thereto, and then the mixture was allowed toreact at 37° C. for 60 minutes. Next, the reaction was stopped byaddition of 0.2 ml of 0.1M Na₂CO₃/NaHCO₃, and the absorbance at 405 nmwas measured by an ELISA analyzer.

(2) Experimental Results

Table 18 below shows the results of measuring of the inhibition (%) ofdegranulation by lactic acid bacteria. As shown in Table 18,Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillusplantarum LC28, Bifidobacterium longum LC67, Bifidobacterium longum LC68and mixtures thereof effectively inhibited the degranulation ofbasophils. Thus, these lactic acid bacteria or mixtures thereof can veryeffectively alleviate allergic atopy, asthma, pharyngitis, chronicdermatitis or the like.

TABLE 18 Drug Degranulation inhibition (%) None 0 LC5 65 LC15 45 LC17 43LC25 48 LC27 52 LC28 54 LC55 38 LC65 42 LC67 65 LC68 61 LC5 + LC67 65LC5 + LC68 60 LC27 + LC67 65 LC27 + LC68 59 LC28 + LC67 62 DSCG(disodiumcromoglycate) 62

5. In Vitro Evaluation of the Anti-Inflammatory and Immune RegulatoryEffects of Lactic Acid Bacteria

(1) Isolation of Macrophages and Measurement of Inflammatory Marker

6-Week-old C57BL/6J male mice (20-23 g) were purchased from RaonBio Co.,Ltd. 2 ml of 4% sterile thioglycolate was administered into theabdominal cavity of each mouse, after 96 hours, the mice wereanesthetized and 8 ml of RPMI 1640 medium was administered into theabdominal cavity of each mouse.

After 5-10 minutes, the RPMI medium (including macrophages) in theabdominal cavity of the mice was taken out, centrifuged at 1000 rpm for10 minutes, and then washed twice with RPMI 1640 medium. The macrophageswere seeded on a 24-well plate at a density of 0.5×10⁶ cells/well andtreated with the test substance lactic acid bacteria and theinflammation inducer LPS (lipopolysaccharide) for 2 hours or 24 hours,and then the supernatant and the cells were collected. In this case, thelactic acid bacteria were used at a concentration of 1×10⁴ CFU/ml fortreatment of the cells. The collected cells were homogenized in buffer(Gibco). Using the collected supernatant, the expression levels ofcytokines such as TNF-α were measured by an ELISA kit. In addition,using the collected cells, the expression levels of p65 (NF-kappa B),p-p65 (phosphor-NF-kappa B) and β-actin were measured by animmunoblotting method. Specifically, 50 μg of the supernatant was takenand electrophoresed on SDS 10% (w/v) polyacrylamide gel for 1 hour and30 minutes. The electrophoresed sample was transferred to anitrocellulose membrane under the conditions of 100 V and 400 mA for 1hour and 10 minutes. The sample-transferred nitrocellulose membrane wasblocked with 5% skim milk for 30 minutes, and then washed three timeswith PBS-Tween for 5 minutes each time, and incubated with a 1:100dilution of primary antibody (Santa Cruz Biotechnology, USA) overnight.Next, the membrane was washed three times for 10 minutes each time, andincubated with a 1:1000 dilution of secondary antibody (Santa CruzBiotechnology, USA) for 1 hour and 20 minutes. Next, the membrane waswashed three times for 15 minutes each time, and it was developed byfluorescence and visualized. The intensity of the developed band wasmeasured, and then inhibition (%) was calculated using the followingequation. In the following equation, the normal group indicates a groupin which macrophages were treated with saline alone; the group treatedwith LPS indicates a group in which macrophages were treated with LPSalone; and the group treated with lactic acid bacteria indicates a groupin which macrophages were treated with both lactic acid bacteria andLPS.

Inhibition (%)=(expression level in group treated with LPS−expressionlevel in group treated with lactic acid bacteria)/(expression level ingroup treated with LPS−expression level in normal group)×100

Table 19 below shows the inhibition of NF-kappa B activation and theinhibition of TNF-α expression when macrophages having inflammationinduced by LPS (lipopolysaccharide) were treated with the lactic acidbacteria. As shown in Table 19 below, Lactobacillus plantarum LC5,Lactobacillus plantarum LC27, Lactobacillus plantarum LC28,Bifidobacterium longum LC67, Bifidobacterium longum LC68 and mixturesthereof effectively inhibited inflammation induced by LPS(lipopolysaccharide).

TABLE 19 Lactic acid bacteria Inhibition (%) of Inhibition (%) of usedfor treatment TNF-α expression p-p65/p65 activation LC5 71 73 LC15 54 55LC17 61 55 LC25 52 65 LC27 70 72 LC28 74 71 LC55 63 62 LC65 65 68 LC6776 77 LC68 75 71 LC5 + LC67 78 72 LC5 + LC68 76 72 LC27 + LC67 81 75LC27 + LC68 77 73 LC28 + LC67 77 73

(2) Isolation of T Cells from Spleen and Measurement of Differentiationinto Th17 Cells or Treg Cells

Spleen was separated from C56BL/6J mice, crushed suitably and suspendedin 10% FCS-containing RPMI 1640 medium, and CD4 T cells were isolatedtherefrom using a CD4 T cell isolation kit (MiltenyiBiotec, BergischGladbach, Germany). The isolated CD4 T cells were seeded in a 12-wellplate at a density of 5×10⁵ cells/well, and anti-CD3 (1 μg/ml,MiltenyiBiotec, Bergisch Gladbach, Germany) and anti-CD28 (1 μg/ml,MiltenyiBiotec, Bergisch Gladbach, Germany) were added thereto, oranti-CD3 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany),anti-CD28 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany),recombinant IL-6 (20 ng/ml, MiltenyiBiotec, Bergisch Gladbach, Germany)and recombinant transforming growth factor beta (1 ng/ml,MiltenyiBiotec, Bergisch Gladbach, Germany) were added. While the cellswere cultured, 1×10³ or 1×10⁵ CFU of the lactic acid bacteria were addedthereto, and the cells were cultured for 4 days. Next, the cells of theculture were stained with anti-FoxP3 or anti-IL-17A antibody, and thedistribution of Th17 cells and Treg cells was analyzed using a FACS(fluorescence-activated cell sorting) system (C6 Flow Cytometer® System,San Jose, Calif., USA).

Table 20 below shows the level of differentiation of T cells (isolatedfrom spleen) into Th17 cells when the T cells were treated withanti-CD3, anti-CD28, IL-6 and TGF-β, and Table 21 below shows the levelof differentiation of T cells (isolated from spleen) into Treg cellswhen the T cells were treated with anti-CD3 and anti-CD28. As shown inTables 20 and 21 below, Lactobacillus plantarum LC5, Lactobacillusplantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longumLC67, Bifidobacterium longum LC68 and mixtures thereof inhibited thedifferentiation of T cells into Th17 cells (T helper 17 cells) andpromoted the differentiation of T cells into Treg cells. These resultssuggest that the lactic acid bacteria or mixtures thereof caneffectively alleviate inflammatory diseases such as colitis orarthritis.

TABLE 20 T-cell treatment method Treatment with anti-CD3, anti-CD28,Treatment with lactic Differentiation (%) IL-6 and TGF-β acid bacteriainto Thl7 cells Not treated Not treated 12.2 Treated Not treated 25.6Treated Treated with LC5 14.2 Treated Treated with LC15 19.6 TreatedTreated with LC17 17.9 Treated Treated with LC25 18.2 Treated Treatedwith LC27 15.1 Treated Treated with LC28 14.9 Treated Treated with LC5518.8 Treated Treated with LC65 17.9 Treated Treated with LC67 15.9Treated Treated with LC68 15.7 Treated Treated with LC5 + LC67 14.2Treated Treated with LC5 + LC68 14.5 Treated Treated with LC27 + LC6713.9 Treated Treated with LC27 + LC68 14.4 Treated Treated with LC28 +LC67 14.1

TABLE 21 T-cell treatment method Differentiation Treatment with anti-CD3Treatment with lactic (%) into Treg and anti-CD28 acid bacteria cellsNot treated Not treated 9.1 Treated Not treated 11.4 Treated Treatedwith LC5 22.9 Treated Treated with LC15 15.8 Treated Treated with LC1716.9 Treated Treated with LC25 18.4 Treated Treated with LC27 21.8Treated Treated with LC28 21.4 Treated Treated with LC55 19.5 TreatedTreated with LC65 19.2 Treated Treated with LC67 21.6 Treated Treatedwith LC68 20.5 Treated Treated with LC5 + LC67 21.8 Treated Treated withLC5 + LC68 21.8 Treated Treated with LC27 + LC67 22.0 Treated Treatedwith LC27 + LC68 21.5 Treated Treated with LC28 + LC67 21.9

6. In Vivo Evaluation of the Anti-Inflammatory and Colitis-AlleviatingEffects of Lactic Acid Bacteria

(1) Test Animals

5-Week-old C57BL/6 male mice (24-27 g) were purchased from OrientBio,and housed under controlled environmental conditions (humidity: 50±10%,temperature: 25±2° C., 12-hr light/12-hr dark cycle), and then used inthe experiment. As feed, standard experimental feed (Samyang, Korea) wasused, and the animals had access to drinking water ad libitum. In allthe experiments, one group consisted of 6 animals.

(2) Colitis Induction by TNBS and Sample Administration

One group of the test animals was used as a normal group, and the testanimals of the other groups were treated with2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce acute colitis.Specifically, the test animals were lightly anesthetized with ether, andthen a mixture solution of 2.5 g of TNBS (2,4,6-trinitrobenzene sulfonicacid) an 100 ml of 50% ethanol was administered into the colon throughthe anal in an amount of 0.1 ml each time by use of a 1-ml round-tipsyringe, and lifted vertically and maintained for 30 seconds, therebyinducing inflammation. On the other hand, the normal group was orallyadministered with 0.1 ml of saline. On the next day, the lactic acidbacteria or the lactic acid bacteria mixture as a test sample wassuspended in saline and administered orally to each mouse in an amountof 2.0×10⁹ CFU, once a day for three days. On the next day following theend of sample administration, the animals were killed with carbondioxide, and a colon portion ranging from the cecum to the site justbefore the anus was dissected and used. Meanwhile, the test animals ofthe normal group were orally administered with saline alone instead ofthe lactic acid bacteria. In addition, the test animals of the negativecontrol group were orally administered with saline alone instead of thelactic acid bacteria after the induction of colitis by TNBS.Furthermore, the test animals of the positive control group were orallyadministered with 50 mg/kg of sulfasalazine, which is a drug fortreating colitis, instead of the lactic acid bacteria.

(3) Macroscopic Analysis of Colon

The length and appearance of the dissected colon were observed, and theappearance was analyzed by scoring according to the criteria (Hollenbachet al., 2005, Criteria for Degree of Colitis) shown in Table 22 below.After complete removal of colon contents, the colon tissue was washedwith saline. A portion of the washed colon tissue was fixed with 4%formaldehyde solution in order to use it as a pathological tissuesample, and the remainder was freeze-stored at −80° C. for molecularbiological analysis.

TABLE 22 Macroscopic score Criteria 0 Any ulcer and inflammation are notfound. 1 Edema without bleeding is found. 2 Ulcer with edema is found. 3Ulcer and inflammation are found at only one site. 4 Ulcer andinflammation are found at two or more sites. 5 Ulcer has an increasedsize of 2 cm or more.

(4) Measurement of Myeloperoxidase (MPO) Activity

100 mg of colon tissue was homogenized in 200 μl of 10 mM potassiumphosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammoniumbromide. The homogenized tissue was centrifuged at 10,000×g and 4° C.for 10 minutes, and the supernatant was collected. 50 μl of thesupernatant was added to 0.95 ml of a reaction solution (containing 1.6mM tetramethyl benzidine and 0.1 mM H₂O₂) and allowed to react at 37°C., and the absorbance at 650 nm was measured at various time pointsduring the reaction. To calculate myeloperoxidase (MPO) activity, 1μmol/ml of peroxide produced by the reaction was used as 1 unit.

(5) Measurement of Inflammatory Marker

Using a Western blotting method, inflammatory markers such as p-p65,p65, iNOS, COX-2 and β-actin were measured.

Specifically, according to the same method as the experiment formeasurement of myeloperoxidase (MPO) activity, a supernatant wasobtained. 50 μg of the supernatant was taken and electrophoresed on SDS10% (w/v) polyacrylamide gel for 1 hour and 30 minutes. Theelectrophoresed sample was transferred to a nitrocellulose membraneunder the conditions of 100 V and 400 mA for 1 hour and 10 minutes. Thesample-transferred nitrocellulose membrane was blocked with 5% skim milkfor 30 minutes, and then washed three times with PBS-Tween for 5 minuteseach time, and incubated with a 1:100 dilution of primary antibody(Santa Cruz Biotechnology, USA) overnight. Next, the membrane was washedthree times for 10 minutes each time, and incubated with a 1:1000dilution of secondary antibody (Santa Cruz Biotechnology, USA) for 1hour and 20 minutes. Next, the membrane was washed three times for 15minutes each time, and it was developed by fluorescence and visualized.

In addition, inflammation-related cytokines such as TNF-α, IL-17, IL-10and the like were measured using an ELISA kit.

(6) Analysis of Immune Regulatory Markers

Dissected colon was washed twice with 2.5 mM EDTA solution. The washedcolon was agitated in RPMI medium containing 1 mg/ml collagenase typeVIII (Sigma) at 30° C. for 20 minutes and was filtered to separate theLamina propria. Next, the Lamina propria was treated with 30-100%percoll solution and centrifuged to separate T cells. The separated Tcells were stained with anti-FoxP3 or anti-IL-17A antibody, and thedistribution of Th17 and Treg cells was analyzed using a FACS(fluorescence-activated cell sorting) system (C6 Flow Cytometer® System,San Jose, Calif., USA).

(7) Experimental Results

Table 23 below shows the effects of the lactic acid bacteria on theweight of the colon, the appearance of the colon, myeloperoxidase (MPO)activity and inflammation-related cytokine contents when the lactic acidbacteria were administered to the model animals having TNBS-inducedacute colitis. As shown in Table 23 below, the model animals havingacute colitis induced by TNBS showed reduced weight, reduced macroscopicscore of the colon, reduced colon length and increased MPO activity.However, when the lactic acid bacteria were administered to the modelanimals having acute colitis induced by TNBS, all these markers wereimproved. In particular, administration of Bifidobacterium longum LC67alone or administration of a mixture of Bifidobacterium longum LC67 andLactobacillus plantarum LC5 showed a very excellent effect on thealleviation of colitis. In addition, the model animals having acutecolitis induced by TNBS showed increased TNF-α and IL-17 levels anddecreased IL-10 levels. However, when the lactic acid bacteria wereadministered to the model animals having acute colitis induced by TNBS,all these markers were improved. In particular, when Bifidobacteriumlongum LC67 was administered alone or a mixture of Bifidobacteriumlongum LC67 and Lactobacillus plantarum LC5 was administered, TNF-α andIL-17 levels greatly decreased, and IL-10 levels greatly increased.

TABLE 23 MPO Weight Macroscopic Colon activity TNF-α IL-17 IL-10 Testgroups gain (g) score length (cm) (μU/mg) (pg/mg) (pg/mg) (pg/mg) Normal0.64 5.9 0.14 0.42 35.1 18.4 61.2 group Negative −2.46 4.2 2.32 1.5495.5 65.2 30.7 control group Group −1.90 4.65 1.30 0.91 75.5 52.8 43.9administered with LC5 Group −1.0 4.56 1.08 0.82 67.2 50.4 44.0administered with LC27 Group −0.28 4.92 0.50 0.43 48.5 38.5 54.6administered with LC67 Group −1.02 4.5 1.34 1.04 54.4 50.5 48.1administered with LC 68 Group −0.3 5.08 0.84 0.42 45.1 37.3 55.3administered with LC5 + LC67 Group −1.15 4.8 1.17 0.78 59.8 45.0 50.0administered with LC27 + LC68 Positive −0.91 4.58 1.43 0.95 58.2 48.545.5 control group

FIG. 22 shows the differentiation patterns of T cells into Th17 cells,which indicate the effect of lactic acid bacteria on model animalshaving acute colitis induced by TNBS, and FIG. 23 shows thedifferentiation patterns of T cells into Treg cells, which indicate theeffect of lactic acid bacteria on model animals having acute colitisinduced by TNBS. In FIGS. 22 and 23, “NOR” indicates a normal group;“TNBS” indicates a negative control group; “LC5” indicates a groupadministered with Lactobacillus plantarum LC5; “LC27” indicates a groupadministered with Lactobacillus plantarum LC27, “LC67” indicates a groupadministered with Bifidobacterium longum LC67; “LC68” indicates a groupadministered with Bifidobacterium longum LC68; “LC5+LC67” indicates agroup administered with a lactic acid bacteria mixture prepared bymixing Lactobacillus plantarum LC5 and Bifidobacterium longum LC67 inthe same amount; “LC27+LC68” indicates a group administered with alactic acid bacteria mixture prepared by mixing Lactobacillus plantarumLC27 and Bifidobacterium longum LC68 in the same amount; and “SS”indicates a group administered with sulfasalazine. As shown in FIGS. 22and 23, in the case of the animals having acute colitis induced by TNBS,the differentiation of T cells into Th17 cells was promoted, and thedifferentiation of T cells into Treg cells was inhibited. However, whenthe lactic acid bacteria were administered to the animals having acutecolitis induced by TNBS, the differentiation of T cells into Th17 cellswas inhibited, and the differentiation of T cells into Treg cells waspromoted. In particular, when Bifidobacterium longum LC67 wasadministered alone or a mixture of Bifidobacterium longum LC67 andLactobacillus plantarum LC5 was administered, the differentiation of Tcells into Th17 cells was significantly inhibited, and thedifferentiation of T cells into Treg cells was significantly promoted.

FIG. 24 shows inflammatory response markers indicating the effect oflactic acid bacteria on model animals having acute colitis induced byTNBS. In FIG. 24, “Nor” indicates a normal group; “T” indicates anegative control group; “LC5” indicates a group administered withLactobacillus plantarum LC5; “LC27” indicates a group administered withLactobacillus plantarum LC27; “LC67” indicates a group administered withBifidobacterium longum LC67; “LC68” indicates a group administered withBifidobacterium longum LC68; “LC5+LC67” indicates a group administeredwith a lactic acid bacteria mixture prepared by mixing Lactobacillusplantarum LC5 and Bifidobacterium longum LC67 in the same amount;“LC27+LC68” indicates a group administered with a lactic acid bacteriamixture prepared by mixing Lactobacillus plantarum LC27 andBifidobacterium longum LC68 in the same amount, and “SS” indicates agroup administered with sulfasalazine. As shown in FIG. 24, in the caseof the model animals having acute colitis induced by TNBS, NF-κB wasactivated (p-p65) and the expression levels of COX-2 and iNOS increased.However, when the lactic acid bacteria were administered, the activationof NF-κB (p-p65) was inhibited, and the expression levels of COX-2 andiNOS also decreased. In particular, administration of Bifidobacteriumlongum LC67 alone or administration of a mixture of Bifidobacteriumlongum LC67 and Lactobacillus plantarum LC5 exhibited excellent effectson the inhibition of NF-κB activation (p-p65) and on the inhibition ofexpression of COX-2 and iNOS.

7. In Vivo Evaluation of the Effect of Lactic Acid Bacteria onAlleviation of Alcohol-Induced Gastric Ulcer

(1) Test Animals

5-Week-old C57BL/6 male mice (24-27 g) were purchased from OrientBio,and housed under controlled environmental conditions (humidity: 50±10%,temperature: 25±2° C., 12-hr light/12-hr dark cycle), and then used inthe experiment. As feed, standard experimental feed (Samyang, Korea) wasused, and the animals had access to drinking water ad libitum. In allthe experiments, one group consisted of 6 animals.

(2) Induction of Gastric Ulcer by Alcohol and Administration of Sample

To one test group, 1×10⁹ CFU of Lactobacillus plantarum LC27 suspendedin saline was orally administered once a day for 3 days. To another testgroup, 1×10⁹ CFU of Bifidobacterium longum LC67 suspended in saline wasorally administered once a day for 3 days. To still another test group,1×10⁹ CFU of a lactic acid bacteria mixture prepared by mixingLactobacillus plantarum LC27 and Bifidobacterium longum LC67 in the sameamount was orally administered once a day for 3 days, after it wassuspended in saline. In addition, to a positive control group,ranitidine, a commercial agent for treating gastric ulcer, was orallyadministered once a day for 3 days in an amount of 50 mg/kg. Inaddition, to a normal group and a negative control group, 0.2 ml ofsaline was orally administered one a day for 3 days. After the samplewas orally administered for 3 days, the test mice were fasted andwater-deprived for 18 hours. On day 4 of the experiment, at 1 hour afteradministration of saline, 0.2 ml of 99% pure ethanol was administeredorally to the mice of all the test groups other than the normal group toinduce gastric ulcer. In addition, to the normal group, 0.2 ml of salinewas administered instead of ethanol.

(3) Measurement of Macroscopic Marker Related to Gastric Injury

3 Hours after administration of ethanol, the test mice were sacrificedand gastric tissue was dissected split longitudinally and washed withPBS (phosphate buffer saline) solution, and then the degree of gastricinjury was observed visually or microscopically and scored (see Park, S.W., Oh, T. Y., Kim, Y. S., Sim, H., et al., Artemisia asiatica extractsprotect against ethanol-induced injury in gastric mucosa of rats. J.Gastroenterol. Hepatol. 2008, 23, 976-984).

(4) Measurement of Myeloperoxidase (MPO) Activity

100 mg of the gastric tissue was homogenized in 200 μl of mM potassiumphosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammoniumbromide. Then, the tissue solution was centrifuged at 10,000×g and 4° C.for 10 minutes, and the supernatant was collected. 50 μl of thesupernatant was added to 0.95 ml of a reaction solution (containing 1.6mM tetramethyl benzidine and 0.1 mM H₂O₂) and allowed to react at 37°C., and the absorbance at 650 nm was measured at various time pointsduring the reaction. To calculate myeloperoxidase (MPO) activity, 1μmol/ml of peroxide produced by the reaction was used as 1 unit.

(5) Measurement of Inflammatory Markers

2 μg of mRNA was isolated from gastric tissue by a Qiagen RNeasy MiniKit and synthesized into cDNA using Takara Prime Script Rtase. Next, theexpression levels of CXCL4 [chemokine (C-X-C motif) ligand 4] and TNF-α(tumor necrosis factor-alpha) were measured using a quantitative realtime polymerase chain reaction (Qiagen thermal cycler, Takara SYBERpremix agent, Thermal cycling conditions: activation of DNA polymerasefor 5 min at 95° C., followed by 40 cycles of amplification for 10 s at95° C. and for 45 s at 60° C.). Table 24 below shows the primersequences used to analyze each cytokine in the quantitative real timepolymerase chain reaction.

TABLE 24 Cytokine to Kind of be analyzed primerPrimer nucleotide sequence TNF-α Forward 5′-CTGTAGCCCACGTCGTAGC-3′Reverse 5′-TTGAGATCCATGCCGTTG-3′ CXCL4 Forward5′-AGTCCTGAGCTGCTGCTTCT-3′ Reverse 5′-GATCTCCATCGCTTTCTTCG-3′

(6) Experimental Results

FIG. 25 depicts images showing the effect of lactic acid bacteria on thestomach mucosa of mice having gastric ulcer induced by ethanol, in thesecond experiment of the present invention; FIG. 26 shows the grossgastric lesion score indicating the effect of lactic acid bacteria onthe stomach mucosa of mice having gastric ulcer induced by ethanol, inthe second experiment of the present invention; FIG. 27 shows the ulcerindex indicating the effect of lactic acid bacteria on the stomachmucosa of mice having gastric ulcer induced by ethanol, in the secondexperiment of the present invention; and FIG. 28 shows the histologicalactivity index indicating the effect of lactic acid bacteria on thestomach mucosa of mice having gastric ulcer induced by ethanol, in thesecond experiment of the present invention. Furthermore, FIG. 29 showsthe myeloperoxidase (MPO) activity indicating the effect of lactic acidbacteria on the stomach mucosa of mice having gastric ulcer induced byethanol, in the second experiment of the present invention. In addition,FIG. 30 shows CXCL4 expression levels indicating the effect of lacticacid bacteria on the stomach mucosa of mice having gastric ulcer inducedby ethanol, in the second experiment of the present invention; and FIG.31 shows TNF-α expression levels indicating the effect of lactic acidbacteria on the stomach mucosa of mice having gastric ulcer induced byethanol, in the second experiment of the present invention. In FIGS. 30and 31, the CXCL4 expression levels and TNF-α expression levels in thetest groups other than the normal group are expressed fold-changesrelative to the expression levels in the normal group. In FIGS. 25 to31, “Nor” indicates a normal group; “Ethanol” indicates a negativecontrol group having ethanol-induced gastric ulcer and administered withsaline as a sample; “Ethanol+Ranitidine” indicates a test group havingethanol-induced gastric ulcer and administered with Ranitidine as asample; “Ethanol+LC27” indicates a test group having ethanol-inducedgastric ulcer and administered with Lactobacillus plantarum LC27 as asample; “Ethanol+LC67” indicates a test group having ethanol-inducedgastric ulcer and administered with Bifidobacterium longum LC67 as asample; and “Ethanol+LC27/LC67” indicates a test group havingethanol-induced gastric ulcer and administered with a lactic acidbacteria mixture, prepared by mixing Lactobacillus plantarum LC27 andBifidobacterium longum LC67 in the same amount, as a sample. As shown inFIGS. 25 to 29, Bifidobacterium longum LC67, Lactobacillus plantarumLC27 or a mixture thereof effectively alleviated the gastric injury orgastric ulcer induced by ethanol. Furthermore, as shown in FIGS. 30 and31, Bifidobacterium longum LC67, Lactobacillus plantarum LC27 or amixture thereof greatly reduced the inflammatory marker levels in themice having ethanol-induced gastric injury or gastric ulcer.

8. In Vivo Evaluation of the Effect of Lactic Acid Bacteria onAlleviation of Alcohol-Induced Liver Injury

(1) Test Animals

5-Week-old C57BL/6 male mice (24-27 g) were purchased from OrientBio,and housed under controlled environmental conditions (humidity: 50±10%,temperature: 25±2° C., 12-hr light/12-hr dark cycle), and then used inthe experiment. As feed, standard experimental feed (Samyang, Korea) wasused, and the animals had access to drinking water ad libitum. In allthe experiments, one group consisted of 6 animals.

(2) Induction of Liver Injury by Alcohol and Administration of Sample

To one test group, 1×10⁹ CFU of Lactobacillus plantarum LC27 suspendedin saline was orally administered once a day for 3 days. To another testgroup, 1×10⁹ CFU of Bifidobacterium longum LC67 suspended in saline wasorally administered once a day for 3 days. To still another test group,1×10⁹ CFU of a lactic acid bacteria mixture prepared by mixingLactobacillus plantarum LC27 and Bifidobacterium longum LC67 in the sameamount was orally administered once a day for 3 days, after it wassuspended in saline. To a positive control group, silymarin, acommercial agent for treating liver injury, was orally administered oncea day for 3 days in an amount of 50 mg/kg. In addition, to a normalgroup and a negative control group, 0.1 ml of saline was orallyadministered once a day for 3 days. 3 hours after 3 days of oraladministration of the sample or saline, ethanol was administeredintraperitoneally to the mice of all the test groups other than thenormal group in an amount of 6 ml/kg in order to induce liver injury. Inaddition, to the normal group, saline in place of ethanol wasadministered intraperitoneally in an amount of 6 ml/kg. Next, the testmice were fasted and water-deprived for 12 hours, and then sacrificed,and blood was taken from the heart.

(3) Measurement of Liver Function Markers and Results

The taken blood was allowed to stand at room temperature for 60 minutesand centrifuged at 3,000 rpm for 15 minutes to separate serum. The GPT(glutamic pyruvate transaminase) and GOT (glutamic oxalacetictransaminase) levels in the separated serum were measured using a bloodassay kit (ALT & AST measurement kit; Asan Pharm. Co., Korea), and theresults of the measurement are shown in Table 25 below. As shown inTable 25 below, Bifidobacterium longum LC67, Lactobacillus plantarumLC27 or a mixture thereof effectively alleviated ethanol-induced liverinjury. In particular, Bifidobacterium longum LC67 showed a bettereffect than silymarin which is a commercial agent for treating liverinjury.

TABLE 25 GOT GPT Test groups (IU/L) (IU/L) Normal group 52.1 42.2Negative control group 107.7 156.3 Group administered with ethanol andLC27 82.3 95.4 Group administered with ethanol and LC67 62.5 65.8 Groupadministered with ethanol and LC27/LC67 71.4 78.3 Group administeredwith ethanol and silymarin 79.5 87.5 * LC27: Lactobacillus plantarumLC27 * LC67: Bifidobacterium longum LC67 * LC27/LC67: lactic acidbacteria mixture prepared by mixing Lactobacillus plantarum LC27 andBifidobacterium longum LC67 in the same amount.

Although the present invention has been described above with referenceto the examples, the scope of the present invention is not limited tothese examples, and various modifications are possible without departingfrom the scope and idea of the present invention. Therefore, the scopeof protection of the present invention should be interpreted to includeall embodiments falling within the appended claims.

1.-16. (canceled)
 17. A method of preventing or treating one or morediseases selected from a group consisting of intestinal damage, liverinjury, allergic disease and inflammatory disease, comprisingadministering a composition comprising Lactobacillus plantarum LC27(accession number: KCCM 11801P), a culture thereof, a lysate thereof oran extract thereof to a subject.
 18. The method according to claim 17,wherein the Lactobacillus plantarum LC27 has one or more characteristicsselected from antioxidant activity, beta-glucuronidase inhibitoryactivity, lipopolysaccharide (LPS) production inhibitory activity andtight junction protein expression-inducing activity.
 19. The methodaccording to claim 17, wherein the Lactobacillus plantarum LC27comprises a 16S rDNA nucleotide sequence represented by SEQ ID NO: 5.20. The method according to claim 17, wherein the composition furthercomprises one or more lactic acid bacteria selected from a groupconsisting of Lactobacillus brevis CH23 (accession number: KCCM 11762P),Bifidobacterium longum CH57 (accession number: KCCM 11764P),Lactobacillus plantarum LC5 (accession number: KCCM 11800P) andBifidobacterium longum LC67 (accession number: KCCM 11802P).
 21. Themethod according to claim 17, wherein the composition further comprisesBifidobacterium longum LC67 (accession number: KCCM 11802P), a culturethereof, a lysate thereof or an extract thereof.
 22. The methodaccording to claim 17, wherein the intestinal damage is intestinalpermeability syndrome.
 23. The method according to claim 17, wherein theliver injury is selected from a group consisting of hepatitis, fattyliver and liver cirrhosis.
 24. The method according to claim 17, whereinthe allergic disease is selected from a group consisting of atopicdermatitis, asthma, pharyngitis and chronic dermatitis.
 25. The methodaccording to claim 17, wherein the inflammatory disease is selected froma group consisting of gastritis, gastric ulcer, colitis and arthritis.